PSMA Binding Antibody and Uses Thereof

ABSTRACT

The present invention provides a novel PSMA binding antibody termed 10B3 and pharmaceutical and diagnostic uses of the antibody 10B3. The PSMA antibody 10B3 does not cross-compete with the state of the art PMSA binding antibody J591 and has a reduced induction of antigen shift compared to J591 and a unique reactivity with squamous cell carcinoma (SCC) cells of different origin.

RELATED APPLICATIONS

This application is a divisional of U.S. Application No. 16/069,656,filed Jul. 12, 2018, which is the U.S. National Stage of InternationalApplication No. PCT/EP2017/050834, filed Jan. 16, 2017, which designatesthe U.S., published in English, and claims priority under 35 U.S.C. §§119 or 365(c) to European Application No. 16151281.9, filed on Jan. 14,2016. The entire teachings of the above applications are incorporatedherein by reference.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in the ST.26 (.XML) format and is hereby incorporated byreference in its entirety.

File name: “ASN-00402 Sequence Listing.XML”; created Sep. 8, 2022;23,485 bytes in size.

FIELD OF THE INVENTION

The present invention provides a novel PSMA binding antibody. The PSMAantibody of the invention does not cross-compete with the state of theart PMSA binding antibody J591 and has a reduced induction of antigenshift compared to J591 and a unique reactivity with squamous cellcarcinoma (SCC) cells of different origin. Further, the presentinvention relates to a bispecific PSMAxCD3 antibody molecule. Thepresent invention also relates methods for producing the antibodymolecule of the invention as well as nucleic acids, vectors, and hostcells. The invention further relates to methods of treating ordiagnosing a disease using a PMSA antibody molecule of the invention.

BACKGROUND

Scientific work starting in the 1980ies has established that bispecificantibodies directed to a tumor associated antigen (TAA) and the T cellreceptor (TCR)/CD3-complex are capable of activating T cells resultingin the lysis of TAA expressing tumor cells by the activated T cells(Staerz et al. Nature 1985, 314:628-631; Perez et al. Nature 1985,316:354-356; Jung et al. Proc Natl Acad Sci USA 1986, 83:4479-4483)Since CD3-antibodies, bound to Fc receptors (FcRs) via their Fc-part,are exceedingly efficient in inducing T cell activation and cytokinerelease as unwanted side effects, it is of paramount importance toconstruct Fc-depleted or -attenuated bispecific TAAxCD3-antibodies inorder to prevent FcR binding and to allow for a target cell restricted-rather than FcR-mediated activation of T cells (Jung et al. ImmunolToday 1988; 9:257-260; Jung et al. Eur J Immunol 1991; 21:2431-2435).

The production of bispecific antibodies meeting this criticalprerequisite in industrial quality and quantity remains a formidablechallenge. Recently, a recombinant, bispecific single chain (bssc)antibody with CD19xCD3-specificity, termed Blinatumomab, hasdemonstrated considerable efficiency in the treatment of patients withALL (Bargou et al. Science 2008, 321:974-977) and has received approvalunder a break through designation by the FDA. Notably, the drug isapplied as continuous 24 hr infusion over several weeks due to its lowserum half-life and rather high toxicity: safely applicable doses areapprox. 30 µg per patient and day which is 10.000 times lower than thoseused for treatment with established monospecific antitumor antibodies(Adams and Weiner. Nat Biotechnol 2005, 23:1147-57). The resulting serumconcentrations of the drug are below 1 ng/ml (Topp et al. J Clin Oncol2011; 29:2493-2498). This severe dose limitation, also observed inearlier clinical trials with different bispecific antibodies (Kroesen etal. Br J Cancer 1994; 70:652-661; Tibben et al. Int J Cancer 1996;66:477-483), is due to off-target T cell activation resulting insystemic cytokine release. Obviously, this phenomenon prevents anoptimal therapeutic activity of bispecific antibodies stimulating theTCR/CD3 complex.

In principle, dose limiting off-target T cell activation and theresulting toxicity problem may be caused by the problems P1 and P2discussed in more detail in the following; low serum half-life isdiscussed as problem P3:

(P1) The TAA targeted by the bispecific antibody is not entirely tumorspecific resulting in antibody mediated T cell activation due to bindingto normal, TAA expressing cells. In a strict sense this is no off targetactivation, since it is induced by antigen expressing target cellsalbeit the “wrong ones”, that is, normal rather than malignant cells.Blinatumomab, the bispecific CD19xCD3-antibody mentioned above,certainly faces this problem since its target antigen CD19 is expressedon normal B lymphocytes. Obviously, the specificity of the targetingantigen for malignant tissue is critical to prevent off-target T cellactivation of this kind. PSMA is a particularly suitable antigen in thisrespect since extensive immunohistologic evaluation has revealed thatthe expression of this antigen on normal tissue is restricted toprostatic epithelium, mammary gland and proximal tubules of the kidney[human protein atlas, http://www.proteinatlas.org]. On malignant tissuethe antigen is abundantly expressed on prostate carcinoma cells and on avariety of other solid tumors, such as colon-, mammary- and pancreaticcarcinoma and glioblastoma (Chang et al. Cancer Res 1999, 59:3192; Rosset al. Cancer Met Rev 2005, 24:521). On these latter tumors, howeverPSMA expression is strictly restricted to the vasculature and spares thetumor cells themselves. Curiously, in prostate carcinoma, the only tumorso far with expression on the tumor cells, the vasculature lacks PSMAexpression in most cases (Chang et al. 1999) so that the optimalsituation, that is expression on the vasculature as well as on the tumorcells themselves, is rarely present P1.1).

Apart from its specificity, another property of the targeting antibodymay be critical for its therapeutic activity: the antibody may cause anantigen shift either by “shedding” or uptake of the antigen into thetarget cell. Antigen uptake is desirable in the case of an immunotoxin,which is a construct comprising an antibody and a toxin that usuallyrequires uptake into the cell to exert its activity. However, ifantibodies are used to recruit immunologic effector cells, antigen shiftby whatever mechanism may hamper the activity of the antibodies. Infact, it has been demonstrated that therapeutic CD20 antibodies induceantigen shift in different lymphoma cells to a variable degree and thatthis phenomenon is, at least in part, responsible for the variabletherapeutic effects of these antibodies (Glennie et al. Mol Immunol.2007; 44:3823). In any case, in the context of T cell activatingbispecific antibodies, it appears desirable to select for targetingantibodies that induce minimal antigen shift (P1.2).

(P2) T cell activation is not -as it should be- target cell restricted,that is, even a monovalent CD3 effector binding site within a bispecificantibody construct is capable of inducing some T cell activation in theabsence of target cells to which the antibody binds with its targetingmoiety. This represents off-target activation in a strict sense, sincecells carrying a target antigen are not required to induce thephenomenon. We have noticed that this phenomenon varies considerably ifdifferent CD3 antibodies in different formats are used and if certainstimulating bystander cells (SBCs), such as lymphoma cells (SKW6.4) orendothelial cells (HUVECs) are added that provide co-stimuli for T cellactivation. Thus, one should select a CD3 moiety inducing minimal“off-target” T cell activation for the construction of bispecificantibodies (P2.1).

In addition to T cell activation induced by genuinely monomeric CD3stimulation, a recent paper suggests an alternative mechanism foroff-target activation involving the targeting part of a bispecificantibody; if this part consists of a single chain fragment that inducesclustering of the effector part of the bispecific antibody on the T cellsurface, tonic signaling may be induced resulting in T cell exhaustion(Long et al. Nat Med 2015; 6:581), that is barely detectable byconventional, short term in vitro assays but severely affects in vivoefficiency. These observations have been made using T cells transfectedwith a chimeric antigen receptor (CAR T cells). Chimeric T cellreceptors comprise single chain antibodies as targeting moieties. It ishighly likely that the results of Long et al. (2015) likewise apply tobispecific antibodies with such a targeting part, since these reagents,once bound to a T cell, are functionally equivalent to a T celltransfected with the corresponding CAR. It is well known in the fieldthat most single chain antibodies have the tendency to form multimersand aggregates (Worn et al. J Mol Biol 2001, 305:989-1010), and thus itis not surprising that all but one of the CARs tested by Long et al.(2015) showed the phenomenon of clustering and tonic CD3 signalingalbeit to a variable degree (Long et al. 2015). The problem outlinedhere (P.2.2) calls for a bispecific format that prevents multimerizationof- and clustering by the targeting part.

(P3) Most bispecific formats suffer from a very low serum half-life (1-3hrs) due to reduced molecular weight and lack of CH3 domains. Thus theprototypical Blinatumomab antibody is applied by continuous 24 hr i.v.infusion over several weeks. The use of whole IgG-based formats withincreased serum half-life, such as the IgGsc depicted in FIG. 1B, hasbeen considered unsuitable because the possibly increased off-targetactivation induced by the bivalent C-terminal CD3 binding moiety.

Based on the above, there is a need in the art for improved antibodymolecules that addresses at least one of the problems outlined above.

SUMMARY OF THE INVENTION

The present invention relates to an antibody molecule or anantigen-binding fragment thereof, capable of binding to human prostatespecific membrane antigen (PSMA), comprising: (i) a heavy chain variabledomain comprising the CDRH1 region set forth in SEQ ID NO: 03(GFTFSDFYMY), the CDRH2 region set forth in SEQ ID NO: 04(TISDGGGYTSYPDSVKG), and the CDRH3 region set forth in SEQ ID NO: 05(GLWLRDALDY) or comprising a CDRH1, CDRH2 or CDRH3 sequence having atleast 75% sequence identity or at least 80% sequence identity with SEQID NO: 03, SEQ ID NO: 04, or SEQ ID NO: 05; and (ii) a light chainvariable domain comprising the CDRL1 region set forth in SEQ ID NO: 06(SASSSISSNYLH), the CDRL2 region set forth in SEQ ID NO: 07 (RTSNLAS),and the CDRL3 region set forth in SEQ ID NO: 08 (QQGSYIPFT) orcomprising a CDRL1, CDRL2 or CDRL3 sequence having at least 75 %sequence identity or at least 80% sequence identity with SEQ ID NO: 06,SEQ ID NO: 07, or SEQ ID NO: 08.

The present invention also relates to an antibody molecule or anantigen-binding fragment thereof, capable of binding to human PSMA thatis able to compete with the binding of an antibody molecule of theinvention or antigen-binding fragment thereof to human PSMA.

The present invention further relates a bispecific antibody moleculecomprising (i) a variable region comprising a heavy chain variabledomain and a light chain variable domain of an PMSA binding antibodymolecule of the invention, wherein said variable region comprises afirst binding site capable of binding to human prostate specificmembrane antigen (PSMA); and (ii) a heavy chain variable region and alight chain variable region of an antibody molecule comprising a secondbinding site.

The present invention further relates to a pharmaceutical compositioncomprising an antibody molecule of the invention or an antigen-bindingfragment thereof.

The present invention further relates to an antibody molecule of theinvention or an antigen-binding fragment thereof for use in thediagnosis or treatment of a disease.

The present invention further relates to an in vitro method ofdiagnosing a disease comprising contacting a sample obtained from asubject with an antibody molecule of the invention or an antigen-bindingfragment thereof.

The present invention further relates to a nucleic acid moleculeencoding an antibody molecule of the invention or an antigen-bindingfragment thereof, a vector comprising said nucleic acid molecule, and ahost cell comprising said nucleic acid molecule or said vector.

The present invention further relates to a method of producing anantibody molecule of the invention or an antigen-binding fragmentthereof, comprising expressing a nucleic acid encoding the antibodymolecule under conditions allowing expression of the nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the detaileddescription when considered in conjunction with the non-limitingexamples and the accompanying drawings, in which:

FIGS. 1A-1C depict various formats of bispecific antibody molecules thathave been used in the present invention. Depicted are bispecificPSMAxCD3 antibodies in the Fabsc-format (FIG. 1A) and IgGsc-format (FIG.1B). In both formats binding of the CH2 domain to Fc-receptors isprevented by defined amino acid modifications. Also depicted is the bssc(bispecific single chain Fv) format (FIG. 1C).

FIG. 2 depicts off-target T cell activation by different PSMAxCD3antibodies. PBMC were incubated with the indicated antibodies in theabsence and presence of SKW6.4 lymphoma cells. After 3 days CD69expression of T cells was analyzed by flow cytometry.

FIGS. 3A-3C show T cell activation, which was assessed by ³H thymidineuptake. In FIG. 3A off-target T cell activation in the absence of targetcells is shown while FIG. 3B depicts, in comparison, on-target T cellactivation with PSMAxCD3 antibodies in the Fabsc-format and contain theCD3 antibodies UCHT1 (NPCU) and OKT3 (NPCO), respectively. In FIG. 3Clysis of PSMA expressing target cells by activated T cells isdemonstrated by an Xelligence cytotoxicity assay.

FIGS. 4A-4F depict multimerization and aggregation of differentbispecific antibody formats. In FIG. 4A and FIG. 4B antibodies withFLT3xCD3-specificity are compared (Fabsc- vs. bssc-format) while inFIGS. 4C-4F those with PSMAxCD3-specificity are compared (Fabsc- vs.IgGsc-format). Gel filtration was performed on Superdex S200 columns.

FIGS. 5A-5C depict the binding of the prior art PSMA-antibody J591 andthe antibody of the invention 10B3 to PSMA-expressing cells. Binding(FIG. 5A), lack of binding competition (FIG. 5B) and shift of the PSMAantigen upon antibody binding (FIG. 5C) was assessed by flow cytometryusing PSMA-transfected Sp2/0 cells. In FIG. 5B it is demonstrated thatchimeric (ch) J591, specifically detected by a goat anti human secondaryantibody, was out-competed by murine (mu) J591 but not murine 10B3.

FIGS. 6A-6D show cryostat sections stained with the PSMA binding priorart antibody J591 and the 10B3 antibody of the invention. In FIG. 6A andFIG. 6B a prostate carcinoma sample was stained with both antibodies inparallel and a polymer system from Zytomed, Berlin, Germany (POLHRP-100)while in FIG. 6C and FIG. 6D a squamous cell carcinoma sample wasstained with the two antibodies again in parallel using the polymersystem from Zytomed. Arrows indicate tumor stroma (Tu) and blood vessels(Ve). Representative results from 9 of 10 prostate cancer samples and 7of 10 squamous cell carcinoma samples are shown. On a variety ofdifferent normal human tissues (obtained from BioCat, Heidelberg,Germany, T6234701-2) the staining pattern of the two antibodies wasidentical with the exception of a faint reactivity of 10B3 withepithelial cells in the skin.

FIG. 7 shows the binding of humanized and mouse 10B3 antibody molecules.Bispecific Fabsc antibody molecules with PSMAxCD3(10B3xOKT3)-specificity containing either the humanized, CDR-grafted(h10B3) variable domains or the mouse (m10B3) antibody variable domainswere incubated with PSMA-expressing 22RV1 cells and analyzed by flowcytometry.

FIG. 8 depicts binding of the CD3-targeting part of different PSMAxCD3antibodies. CD3-positive Jurkat cells were incubated with the indicatedantibodies and analyzed by flow cytometry.

FIG. 9 depicts cytolytic activity of the different PSMA antibodies. PSMAexpressing 22RV1 prostate carcinoma cells were incubated with PBMCs andthe indicated bispecific PSMAxCD3 antibodies at a PBMC:target ratio of5:1. The viability of the adherent target cells was assessed using anXelligence system. Representative results of one out of four differentexperiments with PBMCs of different healthy volunteers are shown.

FIGS. 10A-10D show the amino acid sequence of heavy and light chainvariable regions of murine and humanized 10B3 antibody. FIG. 10A showsthe amino acid sequence of the heavy chain variable region of the murine10B3 antibody (SEQ ID NO: 01). CDR sequences are underlined. FIG. 10Bshows the amino acid sequence of the light chain variable region of themurine 10B3 antibody (SEQ ID NO: 02). CDR sequences are underlined. FIG.10C shows the amino acid sequence of the heavy chain variable region ofhumanized 10B3 antibody in which the CDR loops (CDRH1, CDRH2, and CHDR3)of the heavy chain of the murine antibody 10B3 are grafted onto thevariable domain of the of the heavy chain germ line sequence IGHV3-11*06(SEQ ID NO: 09). CDR sequences are underlined. In addition, the serineresidue that is present at position 49 of the heavy chain germ linesequence IGHV3-11*06 is back-mutated in the variable domain of SEQ IDNO:9 to an alanine that is present in the murine antibody 10B3. Thisalanine residue at position 49 is highlighted in bold and italics inFIG. 10C). FIG. 10D shows the amino acid sequence of the light chainvariable region of humanized 10B3 antibody in which the CDR loops(CDRL1, CDRL2, CDRL3 of the light chain of the antibody 10B3 are graftedonto the variable domain of the human κ light sequence IGKV3-20*02 (SEQID NO: 10). CDR sequences are underlined. In addition, the phenylalaninepresent at sequence position 72 in the variable domain of the humanlight chain sequence of IGKV3-20*02 is back-mutated in the variabledomain of SEQ ID NO: 10 to the tyrosine residue that is present at thissequence position in the murine antibody 10B3. This tyrosine residue atposition 72 is highlighted in bold and italics in FIG. 10D.

FIG. 11 shows the amino acid sequence of the heavy chain of the PSMA(humanized h10B3) X CD3 (humanized hUCHT1) bispecific IgGsc formatantibody molecule (SEQ ID NO: 11). The heavy chain comprises thehumanized heavy chain (HC) variable region of 10B3, an IgG1 CH1 domain,an IgG1 hinge region, a modified IgG1 CH2 domain, an IgG1 CH3 domain,and a humanized CD3 (UCHT1) single chain Fv fragment.

FIG. 12 shows the amino acid sequence of the heavy chain of the PSMA(humanized h10B3) X CD3 (murine OKT3) bispecific Fabsc format antibodymolecule (SEQ ID NO: 12). The heavy chain comprises a humanized HCvariable region of 10B3, an IgG1 CH1 domain, an IgG1 hinge region, amodified IgG1 CH2 domain, the beginning of an IgG1 CH3 domain, and amurine CD3 (OKT3) single chain Fv fragment.

FIG. 13 shows the amino acid sequence of the kappa light chain of thePSMA (humanized h10B3) antibody (SEQ ID NO: 13). This light chaincompletes the heavy chain constructs of SEQ ID NO: 11 and SEQ ID NO: 12to form an h10B3xUCHT1 IgGscand a h10B3xOKT3 Fabsc-molecule,respectively (see FIG. 1 ).

FIGS. 14A-14B show the amino acid sequence of the variable domains ofthe antibody J519, with FIG. 14A showing the amino acid sequence of thevariable domain of the heavy chain (SEQ ID NO: 15) and FIG. 14B showingthe amino acid sequence of the variable domain of the light chain (SEQID NO: 16) of the antibody J519.

FIGS. 15A-15F show the therapeutic effect of the bispecific antibody ofthe invention in vitro. PSMA (humanized h10B3) X CD3 (humanized hUCHT1)bispecific IgGsc format antibody molecule of the invention and controlbispecific antibody (NG2xCD3) was incubated in the presence of PBMC withor without tumor cells.

FIGS. 16A-16D show the in vivo anti-tumor activity of the PSMA(humanized h10B3) X CD3 (humanized hUCHT1) bispecific IgGsc formatantibody molecule of the invention in a mouse model.

FIGS. 17A-17B show the T cell activation and tumor cell growthinhibition of non-PSMA targeting bispecific IgGsc antibodies with UCHT1as anti CD3 specificity.

DETAILED DESCRIPTION

The present invention relates to an antibody, an antibody molecule or anantigen-binding fragment thereof that is capable of binding to humanprostate specific membrane antigen (PSMA). The antibody, antibodymolecule or antigen-binding fragment thereof comprises (i) a heavy chainvariable domain comprising the CDRH1 region set forth in SEQ ID NO: 3(having the amino acid sequence GFTFSDFYMY), the CDRH2 region set forthin SEQ ID NO: 4 (having the amino acid sequence TISDGGGYTSYPDSVKG), andthe CDRH3 region set forth in SEQ ID NO: 5 (having the amino acidsequence GLWLRDALDY) or comprising a CDRH1, CDRH2 or CDRH3 sequencehaving at least 75 % sequence identity or at least 80% sequence identitywith SEQ ID NO: 3, SEQ ID NO:4, or SEQ ID NO: 5. It further comprises(iii) a light chain variable domain comprising the CDRL1 region setforth in SEQ ID NO: 6 (having the amino acid sequence SASSSISSNYLH), theCDRL2 region set forth in SEQ ID NO: 7 (having the amino acid sequenceRTSNLAS), and the CDRL3 region set forth in SEQ ID NO: 8 (having theamino acid sequence QQGSYIPFT) or comprising a CDRL1, CDRL2 or CDRL3sequence having 75 % sequence identity or 80% sequence identity with SEQID NO: 6, SEQ ID NO:7, or SEQ ID NO: 8. Envisioned by the invention isan antibody molecule comprising the CDRH1 region set forth in SEQ ID NO:3, the CDRH2 region set forth in SEQ ID NO: 4, the CDRH3 region setforth in SEQ ID NO: 5, the CDRL1 region set forth in SEQ ID NO: 6, theVLCDL2 region set forth in SEQ ID NO: 7, and the VLCDL3 region set forthin SEQ ID NO: 8. In this context, it is noted that the antibody moleculeof the present invention or antigen binding fragment thereof preferablydoes not compete with the binding of the antibody J591 (Liu et al.,Cancer Res 1997; 57: 3629-34, which is the most highly developedantibody clinically see the review of Akhtar et al “Prostate-SpecificMembrane Antigen-Based Therapeutics”, Advances in Urology Volume 2012(2012), Article ID 973820) to human PSMA. In addition, an antibodymolecule of the present invention or antigen binding fragment thereofmay have a reduced induction of antigen shift when binding to PSMAcompared to J591. It may also exert a unique reactivity with squamouscarcinoma cells of different origin.

The present invention further relates to an antibody, an antibodymolecule or antigen-binding fragment thereof comprises a heavy chainvariable region that comprises the amino acid sequence having a sequenceidentity of at least 90% to the amino acid sequence set forth in SEQ IDNO: 01 or 09. Also encompassed by the invention is an antibody, anantibody molecule or an antigen-binding fragment thereof, comprising alight chain variable region, wherein the light chain variable regioncomprises the amino acid sequence having a sequence identity of at least90 % to the amino acid sequence set forth in SEQ ID NO: 02 or SEQ ID NO:10. Particularly preferred is an antibody, an antibody molecule orantigen-binding fragment thereof comprising a heavy chain variabledomain and a light chain variable domain of the murine anti-PSMAantibody 10B3 (m10B3) as set forth in SEQ ID NO: 01 and 02,respectively. Also preferred is an antibody, an antibody molecule orantigen-binding fragment thereof comprising a heavy chain variabledomain and a light chain variable domain of the humanized anti-PSMAantibody 10B3 (h10B3) as set forth in SEQ ID NO: 09 and 10,respectively.

PSMA is a particularly attractive antigen for antibody mediatedtargeting, and several antibodies directed to the extracellular portionof this protein have been developed. The most advanced reagent, J591(Liu et al., Cancer Res 1997; 57: 3629-34), is currently evaluated inclinical trials, either radiolabeled or coupled to the toxin DM1, aderivative of maytansin, a tubulin inhibiting compound (Ross JS, et al.Cancer Met Rev 2005; 24:521; Akhtar et al, 2012, supra). The PSMAantibody of the invention has an identical reaction pattern with normalhuman tissue. The PSMA antibody of the present invention however differsfrom the antibody J591 in its reaction with squamous carcinoma cells ofdifferent origin. It has been surprisingly found here that in thesetumors, as well as in cancer of the prostate both, the tumor cellsthemselves and the vasculature within and around the tumor are stainedby an PMSA antibody such as the antibody 10B3 that contains the CDRsequences of the heavy and light chain variable domains as depicted inSEQ ID NO:3 to SEQ ID NO: 8 (cf. FIGS. 6A-6D). Squamous carcinomas makeup the majority of cancers arising in the ear nose and throatcompartment, the esophagus and the cervix uteri as well as 20-30% oflung tumors, and PSMA expression on such tumors has not been describedbefore. Thus, the favorable reactivity of the antibody of the presentinvention with these cancers offers extended and improved diagnostic andtreatment options.

The PSMA antibody J591 and the antibody of the present invention differin another important respect: In general, many antibodies induce aprofound antigen shift upon binding to a target cell, a desired propertye.g. for the construction of immunotoxins that require uptake into thecells to exert biological activity. The benchmark PSMA antibody J591,for example, is used for such a purpose (Ross et al. 2005, supra). If,however, recruitment of immunological effector cells is desired, astable expression of the antigen is preferable rather than its rapiduptake into the cell. FIGS. 5A-5C demonstrate that binding of anantibody of the present invention to PSMA transfected Sp2/0 cells iscomparable to that of J591 (FIG. 5A) and that the two antibodies do notcross-compete each other, indicating that they bind to differentepitopes of the PSMA molecule (FIG. 5B). Most importantly, the antibodyof the present invention induces a reduced antigen shift if compared toJ591 (FIG. 5C). In this context, it is however noted that the epitope onPMSA to which the antibody 10B3 binds is not yet known. It is also notedin this respect that the epitope to which the antibody 10B3 binds onsquamous carcinoma cells may not necessarily be the same as the epitopeon PMSA, in particular as PMSA expression has not yet been reported onsquamous carcinoma cells. The epitope or epitopes to which an antibodymolecule of the invention binds on squamous carcinoma cells may thus beonly related to the epitope on PMSA with respect to their amino acidsequence or their confirmation. However, the nature of the respectiveepitope on PMSA or squamous carcinoma cells is not relevant in thepresent invention as long as an antibody molecule of the presentinvention binds to cells expressing PMSA or to squamous carcinoma cellsas described here. It is also noted here that the binding of an antibodymolecule of the present invention to a cell does not necessarily have totrigger a physiological response. Rather, it is sufficient that theantibody of the invention binds to (the epitope present on) a givencell. If, for example, conjugated to a cell-toxic agent such a toxin ora radioactive ligand, the antibody serves, for therapeutic purposes, asdelivery or targeting moiety that brings the cell-toxic agent to thecell on which the cell toxic agent should exercise its cell toxic(cell-killing) activity. Likewise, when used for diagnostic purposes, anantibody of the invention may be conjugated to an imaging moiety thatprovides a detectable signal that can be used for detection of the cellto which the antibody has bound.

The present invention also provides a humanized version of 10B3, whichhas been humanized by CDR grafting, meaning the CDR regions of themurine antibody 10B3 are inserted into the framework region of a heavychain and a light chain of a human antibody. In principle any variablehuman light chain and/or variable heavy chain can serve as scaffold forthe CDR grafting. In one illustrative example of a humanized antibody ofthe invention, the CDR regions of the light chain of the antibody 10B3(that means the CDR loops of SEQ ID NO: 6 to SEQ ID NO: 8) can beinserted into (the variable domain) of the human κ light sequenceIGKV3-20*02 that is deposited in the IMGT/LIGM-database under accessionnumber L37729, see also Ichiyoshi Y., Zhou M., Casali P. A humananti-insulin IgG autoantibody apparently arises through clonal selectionfrom an insulin-specific ‘germ-line’ natural antibody template. Analysisby V gene segment reassortment and site-directed mutagenesis’ J.Immunol. 154(1):226-238 (1995). In another illustrative example of ahumanized antibody of the invention, the CDR regions of the heavy chainof the antibody 10B3 (that means the CDR loops of SEQ ID NO: 3 to SEQ IDNO: 5) can be included into the (variable domains) of the heavy chainsequence IGHV3-11*06 which is deposited in the IMGT/LIGM-database underaccession number AF064919 (See also Watson C.T., et al. Completehaplotype sequence of the human immunoglobulin heavy-chain variable,diversity, and joining genes and characterization of allelic andcopy-number variation. Am. J. Hum. Genet. 92(4):530-546 (2013). In afurther illustrative embodiment of a humanized antibody as describedherein, the CDR loops of the heavy chain of the antibody 10B3 aregrafted onto the variable domain of the heavy chain germ line sequenceIGHV3-11*06 and the CDR loops of the light chain of the antibody 10B3are grafted onto the variable domain of the human κ light sequenceIGKV3-20*02. In order to maintain the binding properties of the parentalmurine antibody 10B3, it may be possible that residues of humanframework are mutated back to the amino acid residue that is present ata particular sequence position of the murine antibody 10B3. In anillustrative example of such a humanized antibody, in the variabledomain of the heavy chain of the human germline sequence of IGHV3-11*06the serine at position 49 was back-mutated to an alanine that is presentin the murine antibody 10B3 (see also FIG. 10C in which the alanineresidue at position 49 is highlighted in bold and italics) while in thevariable domain of the light chain sequence of IGKV3-20*02 thephenylalanine at sequence position 72 of the human germline sequence wasback-mutated to a tyrosine residue that is present at this sequenceposition in the murine antibody 10B3 (see also FIG. 10D in which thetyrosine residue at position 72 is highlighted in bold and italics).Such a humanized antibody, incorporated into a bispecificFabsc-antibody, binds with the same avidity to the PSMA expressing cellline than the mouse parental antibody (cf. FIG. 7 ).

The term “antibody” generally refers to a proteinaceous binding moleculethat is based on an immunoglobulin. Typical examples of such an antibodyare derivatives or functional fragments of an immunoglobulin whichretain the binding specificity. Techniques for the production ofantibodies and antibody fragments are well known in the art. The term“antibody” also includes immunoglobulins (Ig’s) of different classes(i.e. IgA, IgG, IgM, IgD and IgE) and subclasses (such as IgG1, lgG2etc.). As also mentioned above, illustrative examples of an antibodyderivative or molecule include Fab fragments, F(ab′)₂, Fv fragments,single-chain Fv fragments (scFv), diabodies or domain antibodies (HoltLJ et al., Trends Biotechnol. 21(11), 2003, 484-490). The definition ofthe term “antibody” thus also includes embodiments such as chimeric,single chain and humanized antibodies.

An “antibody molecule” as used herein may carry one or more domains thathave a sequence with at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85 %, at least about 90%,at least about 92%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98% or at least about 99% sequence identitywith a corresponding naturally occurring domain of an immunoglobulin M,an immunoglobulin G, an immunoglobulin A, an immunoglobulin D or animmunoglobulin E. It is noted in this regard, the term “about” or“approximately” as used herein means within a deviation of 20%, such aswithin a deviation of 10% or within 5% of a given value or range.

“Percent (%) sequence identity” as used in the present invention meansthe percentage of pair-wise identical residues - following homologyalignment of a sequence of a polypeptide of the present invention with asequence in question - with respect to the number of residues in thelonger of these two sequences. Alignment for purposes of determiningpercent amino acid sequence identity can be achieved in various waysthat are within the skill in the art, for instance, using publicallyavailable computer software such as BLAST, ALIGN, or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximum alignment over the full length of the sequences being compared.The same is true for nucleotide sequences disclosed herein. In thiscontext, the sequence identity of at least 75% or at least 80% asdescribed herein is illustrated with respect for the CDR sequence of anantibody of the invention. Referring first to CDR H1, an antibody of theinvention has a CDRH1 sequence GFTFSDFYMY (SEQ ID NO: 3) or an aminoacid sequence having 80% sequence identity with this sequence. Sincethis CDRH1 sequence has a length of 10 amino acid, 2 of these 10residues can be replaced to have a sequence identity of 80% to SEQ IDNO:3. It is for example possible that the threonine residue at position3 of CDR H1 is replaced by a serine (making a conservative substitution)and the serine residue at position 5 of CDRH1 is replaced by a threonineresidue (meaning also by a conservative substitution). Thus, theresulting sequence GFSFTDFYMY (SEQ ID NO: 14) of CDRH1 which carriesthese two conservative amino acid exchanges relative to SEQ ID NO: 3 hasa sequence identity of 80% to the sequence of SEQ ID NO: 3, while a CDRH1 sequence in which only one of these two conservative substitutionsare made has a sequence identity of 90% with the sequence of SEQ ID NO:3. Similarly, the CDRH2 sequence set forth in SEQ ID NO: 04(TISDGGGYTSYPDSVKG) contains 17 amino acid residues, a sequence identityof 80% allows up to 3 mutations relative to the sequence of SEQ ID NO:04 (since 20% theoretically corresponds to 3.4 different amino acids).For example, the first threonine residue of SEQ ID NO: 4 may be replacedby a serine. Similarly, the CDRH3 region set forth in SEQ ID NO: 05(GLWLRDALDY) has a length of 10 amino acid residues. Thus, a CDRH3sequence that has 80% or 90% sequence identity to SEQ ID NO: 05(GLWLRDALDY) can comprise two amino acid replacements, for example,conservative substitutions, compared to SEQ ID NO: 5. The CDRL1 regionset forth in SEQ ID NO: 06 (SASSSISSNYLH) comprises 12 amino acidresidues. Thus, a CDRL1 sequence that carries one or two amino acidsubstitutions compared to the amino acid sequence of SEQ ID NO: 06 has asequence identity of more than 80 % to the sequence of SEQ ID NO: 06.The CDRL2 region set forth in SEQ ID NO: 07 (RTSNLAS) has a length of 7amino acid residues. Thus, a CDRL2 sequence that contains one amino acidsubstitution compared to the CDRL2 sequence of SEQ ID NO: 7 has asequence identity of 84% to the SEQ ID NO: 07. Finally, the CDRL3 regionset forth in SEQ ID NO: 08 (QQGSYIPFT) has a length of 9 amino acidresidues. Accordingly, a CDRL3 sequence that comprises one substitutedamino acid compared to the sequence of SEQ NO: 08 has a sequenceidentity of 89% to SEQ ID NO: 8 and a CDL3 sequence that comprises twoamino acid substitutions compared to SEQ ID NO: 08 has a sequenceidentity of 78% to the sequence of SEQ ID NO: 08. It is noted here thatfrom the above explanation and the sequences of the CDR regionsdescribed herein, the person skilled in the art will understand that anysequences that has at least 80% sequence identity to the sequence of anyof the CDRH1, CDRH2, CDHL3, CDRL1, CDRL2 and CDRL3 described herein (SEQID NO: 03 to SEQ ID NO: 08) and that is able to bind to bind PMSA andpreferably also to squamous carcinoma cells as described herein isencompassed in the present invention. While the CDR sequence that has atleast 75%, at least 80%, at least 85%, or at least 90 % sequenceidentity to the respective CDR sequence of any of SEQ ID NO: 03 to SEQID NO: 08 comprises preferably one or more conservative mutations, it isalso possible that the deviation to the sequence of any of the six“parental” CDR regions (SEQ ID NO: 3 to SEQ ID NO: 8) of the antibody ofthe invention and thus a sequence identity of 75% or more is due to thepresence of no-conservative mutations in the CDR regions as long as theantibody retains the ability to bind PMSA and preferably also tosquamous carcinoma cells.

An “immunoglobulin” when used herein, is typically a tetramericglycosylated protein composed of two light (L) chains of approximately25 kDa each and two heavy (H) chains of approximately 50 kDa each. Twotypes of light chain, termed lambda and kappa, may be found inimmunoglobulins. Depending on the amino acid sequence of the constantdomain of heavy chains, immunoglobulins can be assigned to five majorclasses: A, D, E, G, and M, and several of these may be further dividedinto subclasses (isotypes), e.g., IgG1, lgG2, IgG3, IgG4, IgA1, andIgA2. An IgM immunoglobulin consists of 5 of the basic heterotetramerunit along with an additional polypeptide called a J chain, and contains10 antigen binding sites, while IgA immunoglobulins contain from 2-5 ofthe basic 4-chain units which can polymerize to form polyvalentassemblages in combination with the J chain. In the case of IgGs, the4-chain unit is generally about 150,000 Daltons.

In the IgG class of immunoglobulins, there are several immunoglobulindomains in the heavy chain. By “immunoglobulin (Ig) domain” herein ismeant a region of an immunoglobulin having a distinct tertiarystructure. In the context of IgG antibodies, the IgG isotypes each havethree CH regions: “CH1” refers to positions 118-220, “CH2” refers topositions 237-340, and “CH3” refers to positions 341-447 according tothe EU index as in Kabat et al. By “hinge” or “hinge region” or“antibody hinge region” or “immunoglobulin hinge region” or “H” hereinis meant the flexible polypeptide comprising the amino acids between thefirst and second constant domains of an antibody. Structurally, the IgGCH1 domain ends at EU position 220, and the IgG CH2 domain begins atresidue EU position 237. Thus for IgG the hinge is herein defined toinclude positions 221 (D221 in IgG1) to 236 (G236 in IgG1), wherein thenumbering is according to the EU index as in Kabat et al. The constantheavy chain, as defined herein, refers to the N-terminus of the CH1domain to the C-terminus of the CH3 domain, thus comprising positions118-447, wherein numbering is according to the EU index.

The term “variable” refers to the portions of the immunoglobulin domainsthat exhibit variability in their sequence and that are involved indetermining the specificity and binding affinity of a particularantibody (i.e., the “variable domain(s)”). Variability is not evenlydistributed throughout the variable domains of antibodies; it isconcentrated in sub-domains of each of the heavy and light chainvariable regions. These sub-domains are called “hypervariable regions”,“HVR,” or “HV,” or “complementarity determining regions” (CDRs). Themore conserved (i.e., non-hypervariable) portions of the variabledomains are called the “framework” regions (FR). The variable domains ofnaturally occurring heavy and light chains each include four FR regions,largely adopting a β-sheet configuration, connected by threehypervariable regions, which form loops connecting, and in some casesforming part of, the β-sheet structure. The hypervariable regions ineach chain are held together in close proximity by the FR and, with thehypervariable regions from the other chain, contribute to the formationof the antigen- binding site (see Kabat et al., see below). Generally,naturally occurring immunoglobulins include six CDRs (see below); threein the VH (H1, H2, H3), and three in the VL (L1, L2, L3). In naturallyoccurring immunoglobulins, H3 and L3 display the most extensivediversity of the six CDRs, and H3 in particular is believed to play aunique role in conferring fine specificity to immunoglobulins. Theconstant domains are not directly involved in antigen binding, butexhibit various effector functions, such as, for example, antibody-dependent, cell-mediated cytotoxicity and complement activation.

The terms “V_(H)” (also referred to as VH) and “V_(L)” (also referred toas VL) are used herein to refer to the heavy chain variable domain andlight chain variable domain respectively of an immunoglobulin. Animmunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions. Thus, theterm “hypervariable region” refers to the amino acid residues of anantibody which are responsible for antigen binding. The hypervariableregion includes amino acid residues from a “Complementarity DeterminingRegion” or “CDR”. There are three heavy chains and three light chainCDRs (or CDR regions) in the variable portion of an immunoglobulin.Thus, “CDRs” as used herein refers to all three heavy chain CDRs (CDRH1,CDRH2 and CDRH3), or all three light chain CDRs (CDRL1, CDRL2 and CDRL3)or both all heavy and all light chain CDRs, if appropriate. Three CDRsmake up the binding character of a light chain variable region and threemake up the binding character of a heavy chain variable region. CDRsdetermine the antigen specificity of an immunoglobulin molecule and areseparated by amino acid sequences that include scaffolding or frameworkregions. The exact definitional CDR boundaries and lengths are subjectto different classification and numbering systems. The structure andprotein folding of the antibody may mean that other residues areconsidered part of the antigen binding region and would be understood tobe so by a skilled person. CDRs provide the majority of contact residuesfor the binding of the immunoglobulin to the antigen or epitope.

CDR3 is typically the greatest source of molecular diversity within theantibody-binding site. H3, for example, can be as short as two aminoacid residues or greater than 26 amino acids. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known in the art. For a review of the antibody structure, seeAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds.Harlow et al., 1988. One of skill in the art will recognize that eachsubunit structure, e.g., a CH, VH, CL, VL, CDR, FR structure, includesactive fragments, e.g., the portion of the VH, VL, or CDR subunit bindsto the antigen, i.e., the antigen-binding fragment, or, e.g., theportion of the CH subunit that binds to and/or activates, e.g., an Fcreceptor and/or complement. The CDRs typically refer to the Kabat CDRs,as described in Sequences of Proteins of immunological Interest, USDepartment of Health and Human Services (1991), eds. Kabat et al.Another standard for characterizing the antigen binding site is to referto the hypervariable loops as described by Chothia. See, e.g., Chothia,et al. (1992; J. MoI. Biol. 227:799-817; and Tomlinson et al. (1995)EMBO J. 14:4628-4638. Still another standard is the AbM definition usedby Oxford Molecular’s AbM antibody modelling software. See, generally,e.g., Protein Sequence and Structure Analysis of Antibody VariableDomains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. andKontermann, R., Springer-Verlag, Heidelberg). Embodiments described withrespect to Kabat CDRs can alternatively be implemented using similardescribed relationships with respect to Chothia hypervariable loops orto the AbM-defined loops.

The corresponding immunoglobulin mu heavy chain, gamma heavy chain,alpha heavy chain, delta heavy chain, epsilon heavy chain, lambda lightchain or kappa light chain may be of any species, such as a mammalianspecies, including a rodent species, an amphibian, e.g. of the subclassLissamphibia that includes e.g. frogs, toads, salamanders or newts or aninvertebrate species. Examples of mammals include, but are not limitedto, a rat, a mouse, a rabbit, a guinea pig, a squirrel, a hamster, ahedgehog, a platypus, an American pika, an armadillo, a dog, a lemur, agoat, a pig, a cow, an opossum, a horse, a bat, a woodchuck, anorang-utan, a rhesus monkey, a woolly monkey, a macaque, a chimpanzee, atamarin (saguinus oedipus), a marmoset or a human.

As mentioned herein an immunoglobulin is typically a glycoprotein thatincludes at least two heavy (H) chains and two light (L) chains linkedby disulfide bonds, or an antigen binding portion thereof. Each heavychain has a heavy chain variable region (abbreviated herein as V_(H))and a heavy chain constant region. In some embodiments the heavy chainconstant region includes three domains, C_(H1), C_(H2) and C_(H3). Eachlight chain has a light chain variable region (abbreviated herein asV_(L)) and a light chain constant region. The light chain constantregion includes one domain, C_(L). The V_(H) and V_(L) regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). The CDRs containmost of the residues responsible for specific interactions of theantibody with the antigen. Each V_(H) and V_(L) has three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anepitope of an antigen.

“Framework Region” or “FR” residues are those variable domain residuesother than the hypervariable region. The sequences of the frameworkregions of different light or heavy chains are relatively conservedwithin a species. Thus, a “human framework region” is a framework regionthat is substantially identical (about 85% or more, usually 90-95% ormore) to the framework region of a naturally occurring humanimmunoglobulin. The framework region of an antibody, that is thecombined framework regions of the constituent light and heavy chains,serves to position and align the CDR’s. The CDR’s are primarilyresponsible for binding to an epitope of an antigen.

The terms “Fab”, “Fab region”, “Fab portion” or “Fab fragment” areunderstood to define a polypeptide that includes a V_(H), a C_(H)1, aV_(L), and a C_(L) immunoglobulin domain. Fab may refer to this regionin isolation, or this region in the context of an antibody molecule, aswell as a full length immunoglobulin or immunoglobulin fragment.Typically a Fab region contains an entire light chain of an antibody. AFab region can be taken to define “an arm” of an immunoglobulinmolecule. It contains the epitope-binding portion of that Ig. The Fabregion of a naturally occurring immunoglobulin can be obtained as aproteolytic fragment by a papain-digestion. A “F(ab′)₂ portion” is theproteolytic fragment of a pepsin-digested immunoglobulin. A “Fab′portion” is the product resulting from reducing the disulfide bonds ofan F(ab′)₂ portion. As used herein the terms “Fab”, “Fab region”, “Fabportion” or “Fab fragment” may further include a hinge region thatdefines the C-terminal end of the antibody arm. This hinge regioncorresponds to the hinge region found C-terminally of the CH1 domainwithin a full length immunoglobulin at which the arms of the antibodymolecule can be taken to define a Y. The term hinge region is used inthe art because an immunoglobulin has some flexibility at this region. A“Fab heavy chain” as used herein is understood as that portion orpolypeptide of the Fab fragment that comprises a V_(H) and a C_(H)1,whereas a “Fab light chain” as used herein is understood as that portionor polypeptide of the Fab fragment that comprises a V_(L), and a C_(L).

The term “Fc region” or “Fc fragment” is used herein to define aC-terminal region of an immunoglobulin heavy chain, includingnative-sequence Fc regions and variant Fc regions. The Fc part mediatesthe effector function of antibodies, e.g. the activation of thecomplement system and of Fc-receptor bearing immune effector cells, suchas NK cells. In human IgG molecules, the Fc region is generated bypapain cleavage N-terminal to Cys226. Although the boundaries of the Fcregion of an immunoglobulin heavy chain might vary, the human IgGheavy-chain Fc region is usually defined to stretch from an amino acidresidue at position Cys226, or from Pro230, to the carboxyl-terminusthereof. The C-terminal lysine (residue 447 according to the EUnumbering system) of the Fc region may be removed, for example, duringproduction or purification of the antibody molecule, or by recombinantlyengineering the nucleic acid encoding a heavy chain of the antibodymolecule. Native-sequence Fc regions include mammalian, e.g. human ormurine, IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4. The Fc region containstwo or three constant domains, depending on the class of the antibody.In embodiments where the immunoglobulin is an IgG the Fc region has aCH2 and a CH3 domain.

The term “single-chain variable fragment” (scFv) is used herein todefine an antibody fragment, in which the variable regions of the heavy(VH) and light chains (VL) of a immunoglobulin are fused together, whichare connected with a short linker peptide of ten to about 25 aminoacids. The linker is usually rich in glycine for flexibility, as well asserine or threonine for solubility, and can either connect theN-terminus of the VH with the C-terminus of the VL, or connect theN-terminus of the VL with the C-terminus of the VH. The scFv fragmentretains a specific antigen binding site but lacks constant domains ofimmunoglobulins.

The term “epitope”, also known as the “antigenic determinant”, refers tothe portion of an antigen to which an antibody or T-cell receptorspecifically binds, thereby forming a complex. Thus, the term “epitope”includes any molecule or protein determinant capable of specific bindingto an immunoglobulin or T-cell receptor. The binding site(s) (paratope)of an antibody molecule described herein may specifically bindto/interact with conformational or continuous epitopes, which are uniquefor the target structure. Epitopic determinants usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics. In someembodiments, epitope determinants include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. With regard to polypeptide antigens a conformational ordiscontinuous epitope is characterized by the presence of two or morediscrete amino acid residues, separated in the primary sequence, butassembling to a consistent structure on the surface of the molecule whenthe polypeptide folds into the native protein/antigen (SeIa, M., Science(1969) 166, 1365-1374; Laver, W.G, et al. Cell (1990) 61, 553-556). Thetwo or more discrete amino acid residues contributing to the epitope maybe present on separate sections of one or more polypeptide chain(s).These residues come together on the surface of the molecule when thepolypeptide chain(s) fold(s) into a three-dimensional structure toconstitute the epitope. In contrast, a continuous or linear epitopeconsists of two or more discrete amino acid residues, which are presentin a single linear segment of a polypeptide chain.

The term “specific” in this context, or “specifically binding”, alsoused as “directed to”, means in accordance with this invention that theantibody or immune receptor fragment is capable of specificallyinteracting with and/or binding to a specific antigen or ligand or a setof specific antigens or ligands but does not essentially bind to otherantigens or ligands. Such binding may be exemplified by the specificityof a “lock-and-key-principle”. Antibodies are said to “bind to the sameepitope” if the antibodies cross-compete so that only one antibody canbind to the epitope at a given point of time, i.e. one antibody preventsthe binding or modulating effect of the other.

Typically, binding is considered specific when the binding affinity ishigher than 10⁻⁶ M or 10⁻⁷ M. In particular, binding is consideredspecific when binding affinity is about 10⁻⁸ to 10⁻¹¹ M (K_(D)), or ofabout 10⁻⁹ to 10⁻¹¹ M or even higher. If necessary, nonspecific bindingof a binding site can be reduced without substantially affectingspecific binding by varying the binding conditions.

The term “isolated antibody molecule” as used herein refers to anantibody molecule that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are matter that would interferewith diagnostic or therapeutic uses for the antibody, and may includeenzymes, hormones, and other proteinaceous or nonproteinaceous solutes.In some embodiments the antibody molecule is purified to greater than95% by weight of antibody as determined by the Lowry method, such asmore than 99% by weight. In some embodiments the antibody is purified tohomogeneity as judged by SDS-PAGE under reducing or nonreducingconditions using Coomassie blue or, preferably, silver stain. Anisolated antibody molecule may in some embodiments be present withinrecombinant cells with one or more component(s) of the antibody’snatural environment not being present. Typically an isolated antibody isprepared by at least one purification step.

A (recombinant) antibody molecule of the invention that binds to PMSAand/or squamous cancer cells as described herein may be used in anysuitable recombinant antibody format, for example as an Fv fragment, ascFv, a univalent antibody lacking a hinge region, a minibody, a Fabfragment, a Fab′ fragment, a F(ab′)₂ fragment. A recombinant antibodymolecule of the invention may also comprise constant domains (regions)such a human IgG constant region, a CH1 domain (as Fab fragments do)and/ or an entire Fc region. Alternatively, an antibody molecule of theinvention may also be a full length (whole) antibody.

There are a number of possible mechanisms by which antibodies mediatecellular effects, including anti-proliferation via blockage of neededgrowth pathways, intracellular signaling leading to apoptosis, enhanceddown regulation and/or turnover of receptors, complement-dependentcytotoxicity (CDC), antibody-dependent cell- mediated cytotoxicity(ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) andpromotion of an adaptive immune response (Cragg et al , 1999, Curr OpinImmunol 11 541-547, Glennie et al, 2000, Immunol Today 21 403-410).Antibody efficacy may be due to a combination of these mechanisms, andtheir relative importance in clinical therapy for oncology appears to becancer dependent.

The importance of FcyR-mediated effector functions for the activity ofsome antibodies has been demonstrated in mice (Clynes et al , 1998, ProcNatl Acad Sci U S A 95 652-656, Clynes et al , 2000, Nat Med 6443-446,), and from observed correlations between clinical efficacy inhumans and their allotype of high (V158) or low (F158) affinitypolymorphic forms of FcγRIIIa (Cartron et al , 2002, Blood 99 754-758,Weng & Levy, 2003, Journal of Clinical Oncology, 21 3940-3947). Togetherthese data suggest that an antibody that is optimized for binding tocertain FcγRs may better mediate effector functions, and thereby destroytarget cells more effectively in patients. Thus a promising means forenhancing the anti-tumor potency of antibodies is via enhancement oftheir ability to mediate cytotoxic effector functions such as ADCC,ADCP, and CDC Additionally, antibodies can mediate anti-tumor mechanismvia growth inhibitory or apoptotic signaling that may occur when anantibody binds to its target on tumor cells. Such signaling may bepotentiated when antibodies are presented to tumor cells bound to immunecells via FcγR. Therefore increased affinity of antibodies to FcyRs mayresult in enhanced antiproliferative effects.

Some success has been achieved at modifying antibodies with selectivelyenhanced binding to FcγRs to provide enhanced effector function.Antibody engineering for optimized effector function has been achievedusing amino acid modifications (see for example U.S. Pat. Application US2004-0132101 or U.S. Pat. Application 2006-0024298.

The present invention therefore also contemplates that the antibodymolecule of the invention or antigen binding fragment thereof ismodified to have enhanced affinity to the FcγRIIIa receptor or hasenhanced ADCC effector function as compared to the parent antibody. Oneway to achieve the enhanced ADCC is by introducing the amino acidsubstitutions 239D and 332E in the CH2 domain of the Fc part of theantibody molecule, for example into the murine or humanized 10B3antibody. The cell killing activity of these antibodies may then besignificantly increased or even detected and generated for the firsttime. In one embodiment, the amino acid substitutions are S239D andI332E.

An antibody molecule of the invention is capable of binding to humanPSMA. The term “Prostate Specific Membrane Antigen” or “PSMA” are usedinterchangeably herein, and include variants, isoforms and specieshomologs of human PSMA. PSMA is also designated GlutamatcarboxypeptidaseII, NAALADase I = N-Acetyl-L-aspartyl-L-glutamatpeptidase I,Folathydrolase I (FOLH1). Human PSMA has the UniProt accession numberQ04609 (version 175 of 9 Dec. 2015). Accordingly, antibodies of theinvention may, in certain cases, cross-react with PSMA from speciesother than human, or other proteins which are structurally related tohuman PSMA (e.g. human PSMA homologs). As mentioned before, a preferredembodiment of the present invention is the antibody 10B3 or a humanizedversion thereof. However, also other antibody molecules that bind to thesame epitope as 10B3 are within the scope of the invention.

To determine the epitope, standard epitope mapping methods known in theart may be used. For example, fragments (peptides) of PMSA (e.g.synthetic peptides) that bind the antibody can be used to determinewhether a candidate antibody or antigen-binding fragment thereof bindsthe same epitope. For linear epitopes, overlapping peptides of a definedlength (e.g., 8 or more amino acids) are synthesized. The peptides canbe offset by 1 amino acid, such that a series of peptides covering every8 amino acid fragment of the PSMA protein sequence are prepared. Fewerpeptides can be prepared by using larger offsets, e.g., 2 or 3 aminoacids. In addition, longer peptides (e.g., 9-, 10- or 11-mers) can besynthesized. Binding of peptides to antibodies or antigen-bindingfragments can be determined using standard methodologies includingsurface plasmon resonance (BIACORE) and ELISA assays. For examination ofconformational epitopes, larger PSMA fragments can be used. Othermethods that use mass spectrometry to define conformational epitopeshave been described and can be (see, e.g., Baerga-Ortiz et al., ProteinScience 11:1300-1308, 2002 and references cited therein). Still othermethods for epitope determination are provided in standard laboratoryreference works, such as Unit 6.8 (“Phage Display Selection and Analysisof B-cell Epitopes”) and Unit 9.8 (“Identification of AntigenicDeterminants Using Synthetic Peptide Combinatorial Libraries”) ofCurrent Protocols in Immunology, Coligan et al., eds., John Wiley &Sons. Epitopes can be confirmed by introducing point mutations ordeletions into a known epitope, and then testing binding with one ormore antibodies or antigen-binding fragments to determine whichmutations reduce binding of the antibodies or antigen-binding fragments.

Also within the scope of the invention are antibody molecules thatcompete with an antibody molecule of the invention, such as 10B3, forbinding to PSMA, e.g. to competitively inhibit binding of 10B3 to PSMA.To determine competitive inhibition, a variety of assays known to one ofordinary skill in the art can be employed. For example,cross-competition assays can be used to determine if an antibody orantigen-binding fragment thereof competitively inhibits binding to PSMAby another antibody or antigen-binding fragment thereof. These includecell-based methods employing flow cytometry or solid phase bindinganalysis. Other assays that evaluate the ability of antibodies orantigen-binding fragments thereof to cross-compete for PSMA moleculesthat are not expressed on the surface of cells, in solid phase or insolution phase, also can be used. An assay by which cross-competitioncan be tested is for example given in Example 10.

As mentioned herein the invention encompasses antibodies that have areduced antigen shift compared to J591 when binding to PSMA. Such areduced antigen shift may for example be assessed using the methodessentially described in Example 10. In a preferred embodiment, such areduced antigen shift is detected in PMSA-transfected Sp2/0 cells.

An antibody molecule according to the invention may have two chains, ashorter chain, which may in some embodiments be a light chain, and amain chain, which may in some embodiments also be addressed as the heavychain. The antibody molecule is usually a dimer of these two chains.

An antibody molecule of the invention may preferably be a bispecificantibody molecule. The bispecific antibody molecule may comprise (i) avariable region comprising a heavy chain variable domain and a lightchain variable domain as defined in any one of the preceding claims,wherein said variable region comprises a first binding site capable ofbinding to human prostate specific membrane antigen (PSMA) and (ii) aheavy chain variable region and a light chain variable region of anantibody molecule comprising a second binding site. It is understoodthat the binding site for PSMA is preferably a binding site of aPSMA-binding antibody of the invention described herein.

A “bispecific” or “bifunctional” antibody molecule is an antibodymolecule that has two different epitope/antigen binding sites, andaccordingly has binding specificities for two different target epitopes.These two epitopes may be epitopes of the same antigen or of differentantigens. In contrast thereto a “bivalent antibody” may have bindingsites of identical antigenic specificity.

A “bispecific antibody” may be an antibody molecule that binds oneantigen or epitope with one of two or more binding arms, defined by afirst pair of heavy and light chain or of main and shorter/smallerchain, and binds a different antigen or epitope on a second arm, definedby a second pair of heavy and light chain or of main and smaller chain.Such an embodiment of a bispecific antibody has two distinct antigenbinding arms, in both specificity and CDR sequences. Typically, abispecific antibody is monovalent for each antigen it binds to, that is,it binds with only one arm to the respective antigen or epitope.However, bispecific antibodies can also be dimerized or multimerized.For example, in the dimeric IgGsc format as described herein, theantibody may have two binding sites for each antigen. A bispecificantibody may be a hybrid antibody molecule, which may have a firstbinding region that is defined by a first light chain variable regionand a first heavy chain variable region, and a second binding regionthat is defined by a second light chain variable region and a secondheavy chain variable region. It is envisioned by the invention that oneof these binding regions may be defined by a heavy/light chain pair. Inthe context of the present invention the bispecific antibody moleculemay have a first binding site, defined by variable regions of a mainchain and a smaller chain, and a second, different binding site definedby a variable region of a scFv fragment that is included in the mainchain of the antibody molecule.

Methods of making a bispecific antibody molecule are known in the art,e.g. chemical conjugation of two different monoclonal antibodies or forexample, also chemical conjugation of two antibody fragments, forexample, of two Fab fragments. Alternatively, bispecific antibodymolecules are made by quadroma technology, that is by fusion of thehybridomas producing the parental antibodies. Because of the randomassortment of H and L chains, a potential mixture of ten differentantibody structures are produced of which only one has the desiredbinding specificity.

The bispecific antibody molecule of the invention can act as amonoclonal antibody (MAb) with respect to each target. In someembodiments the antibody is chimeric, humanized or fully human. Abispecific antibody molecule may for example be a bispecific tandemsingle chain Fv, a bispecific Fab₂, or a bispecific diabody.

On the basis of the domains included in an antibody molecule of theinvention the bispecific antibody molecule of the invention may comprisea Fab fragment, which may generally include a hinge region, a CH2 domainand a single chain Fv fragment. Such bispecific antibody molecules aretermed “Fabsc”-antibody molecules and have been described for the firsttime in International patent application WO 2013/092001. Morespecifically, a “Fabsc” format antibody molecule as used here typicallyrefers to a bispecific antibody molecule of the invention having a Fabfragment, which generally includes a hinge region, which is at theC-terminus of the Fab fragment linked to the N-terminus of a CH2 domain,of which the C-terminus is in turn linked to the N-terminus of a scFvfragment. Such a “Fabsc” does not or does not essentially comprise a CH3domain. In this context, “not comprising” or “not essentiallycomprising” means that the antibody molecule does not comprise a fulllength CH3 domain. It preferably means that the antibody moleculecomprises 10 or less, preferably 5 or less, preferably 3 or even lessamino acids of the CH3 domain. An illustrative example for a Fabscformat antibody molecule is shown in FIG. 1A, another illustrativeexample for a Fabsc format antibody molecule is show in FIG. 12 . Inillustrative embodiments (cf. also FIG. 1A in this respect, an Fabscantibody molecule of the invention may comprise a CH2 domain that lacksis ability to dimerize by the disulphide bonds that are formed by thecysteine residue at sequence position 226 of the hinge region and/or thecysteine residue at sequence position 229 of one of the hinge domains,according to the Kabat numbering [EU-Index]. Thus, in these embodiments,the cysteine residues at sequence position 226 and/or sequence position229 is either removed or replaced, for example, by a serine residue. Inaddition, or alternatively, an “Fabsc” antibody molecule of theinvention may also have an “Fc-attenuated” CH2 domain (that includes thehinge region). This “Fc-attenuation” is achieved by deleting and/ orsubstituting (mutating) at least one of selected amino acid residues inthe CH2 domain that are able to mediate binding to an Fc-receptor. Inillustrative embodiments, the at least one amino acid residue of thehinge region or the CH2 domain that is able to mediate binding to Fcreceptors and that is lacking or mutated, is selected from the groupconsisting of sequence position 228, 230, 231, 232, 233, 234, 235, 236,237, 238, 265, 297, 327, and 330 (numbering of sequence positionsaccording to the EU-index). In illustrative example, such anFc-attenuated antibody molecule may contain at least one mutationselected from the group consisting of a deletion of amino acid 228, adeletion of amino acid 229, a deletion of amino acid 230, a deletion ofamino acid 231, a deletion of amino acid 232, a deletion of amino acid233, a substitution Glu233→Pro, a substitution Leu234→Val, a deletion ofamino acid 234, a substitution Leu235→Ala, a deletion of amino acid 235,a deletion of amino acid 236, a deletion of amino acid 237, a deletionof amino acid 238, a substitution Asp265→Gly, a substitution Asn297→Gln,a substitution Ala327→Gln, and a substitution Ala330→Ser (numbering ofsequence positions according to the EU-index, see in respect, forexample, also Fig. 1O and Fig. 1P of International patent application WO2013/092001). In the case of bispecific antibodies that activate Tcells, e.g. against tumor cells, Fc-attenuation may be desired toprevent binding of the antibodies to Fc-receptor carrying cells whichmay lead to undesirable off-target activation of T cells.

In accordance with the publication of Coloma and Morrison (NatBiotechnol 15:159-63, 1997), a bispecific antibody molecule of theinvention may also have a CH3 domain, generally arranged C-terminally ofthe CH2 domain. Such a molecule is also referred to herein as an “IgGsc”format antibody molecule and means a bispecific antibody molecule of theinvention having a Fab fragment, which generally includes a hingeregion, which is at the C-terminus of the Fab fragment typically linkedto the N-terminus of a CH2 domain, of which the C-terminus is in turntypically linked to the N-terminus of a CH3 domain, of which theC-terminus is in turn typically linked to the N-terminus of a scFvfragment. An illustrative example of an IgGsc format antibody moleculeis shown in FIG. 1B, heavy and light chain sequences for such a moleculeare molecule shown in FIGS. 11 and 13 , respectively.

The antibody formats Fabsc and IgGsc have both in common that theN-terminal targeting part consists of “physiological” Fab- or Fab₂regions, respectively, thereby avoiding the use of single chain moietiesin this part of the molecule. If these formats are to be used for targetcell restricted T cell activation, attenuation of Fc receptor (FcR)binding may be employed (if wanted or required) to prevent FcR mediatedactivation. This can be achieved e.g. by introduction of defined andwell known mutations in the CH2 domain of the molecule as described inabove and also in International patent application WO 2013/092001 and inArmour et al. Eur J Immunol 1999; 29:2613. Accordingly, also an IgGscantibody molecule of the invention may have a CH2 domain (including thehinge region) in which at least one amino acid residue of the hingeregion or the CH2 domain that is able to mediate binding to Fc receptorsis lacking or mutated. As explained above, this residue in the CH2 andhinge region, respectively, may be selected from the group consisting ofsequence position 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 265,297, 327, and 330 (numbering of sequence positions according to theEU-index). However, due to the presence of the CH3 domain in the IgGscmolecule, two individual molecules will (spontaneously) homodimerize viathe CH3 domain to form a tetravalent molecule (see again FIG. 1B in thisrespect). Thus, it is not necessary to delete or mutate the cysteineresidues at sequence position 226 and/or sequence position 229 of thehinge region. Thus, such a tetrameric IgGsc antibody molecule of theinvention may have a cysteine residue at sequence position 226 and/or atsequence position 229 of one of the respective hinge domain, in linewith the Kabat numbering [EU-Index].

In line with the above disclosure of the bispecific antibody moleculesthat contain a set of CDR regions (for example, the CDR sequences of SEQID NO: 3 to SEQ ID NO:8 or a sequence with 80% sequence identity) thatmediate PMSA binding and/or binding to squamous cancer cells, theantibody molecule of the present invention may comprise a second bindingsite that specifically binds to a receptor on an immune cell such as a Tcell or an NK cells. This receptor present on the immune cell may be areceptor that is capable of activating the immune cell or of stimulatingan immune response of the immune cell. The evoked immune response maypreferably be a cytotoxic immune response. Such a suitable receptor may,for example, be CD3, the antigen specific T cell receptor (TCR), CD28,CD16, NKG2D, Ox40, 4-1 BB, CD2, CD5, programmed cell death protein 1(PD-1) and CD95. Particularly preferred is an antibody molecule in whichthe second binding site binds to CD3, TCR or CD16. Most preferred is anantibody molecule, in which the second binding site specifically bindsto CD3. A preferred antibody molecule comprises a second binding sitethat corresponds to the antigen binding site of the anti-CD3 antibodyOKT3. The VH and VL sequences of an OKT3 antibody are depicted in FIG.12 . The amino acid sequence of the variable domain of the heavy chainand of the variable domain of the light chain of the antibody OKT3 are,for example, also described in Arakawa et al J. Biochem. 120, 657-662(1996) and International Patent Application WO 2015/158868 (see SEQ IDNOS: 17 and 18 in the Sequence Listings of WO 2015/158868). Anotherpreferred antibody molecule comprises a second binding site thatcorresponds to the antigen binding site of the anti-CD3 antibody UCHT1.The VH and VL sequences of a humanized UCHT1 antibody are depicted inFIG. 11 and, for example, also described in International PatentApplication WO 2013/092001. Other examples of CD3 binding antibodymolecules that can be used in the present invention include the antibodymolecules described in European Patent 2 155 783 B1 or European PatentEP 2 155 788 B1 that are capable of binding to an epitope of human andCallithrix jacchus, Saguinus oedipus or Saimiri sciureus CD3ε chain.

Accordingly, the bispecific antibody molecule of the invention may be abispecific antibody molecule such as a Fabsc- and IgGsc-molecule thatcomprises a Fab fragment and a scFv fragment as described herein. Inthis molecule the first binding site may bind to PSMA and may becomprised in a Fab fragment as described herein and the second bindingsite (that may bind to an immune receptor) may be comprised in a scFvfragment. Alternatively, the first binding site that binds to PMSA iscomprised in a single chain Fv fragment and the second binding site(that may bind to an immune receptor) is comprised in a Fab fragment.

In some embodiments, the bispecific antibody molecule of the inventiondoes not by itself activate the immune cell, e.g. the T cell, uponbinding, such as binding to CD3. Instead, only when both binding sites,e.g. the PMSA-specific binding sited and the CD3 specific binding siteare bound to the receptor on the T cell and to PMSA on the target cell,the former may cross-link the activating receptor, triggering theeffector cells to kill the specific target cell. Standard functionalassays to evaluate the target cell -killing capability by lymphocytes inthe presence and absence of an bispecific antibody molecule of theinvention can be set up to assess and/or screen for the ability of theantibody molecule to activate the receptor to which it binds.

Without wishing to be bound to theory, it is generally held thatbispecific CD3-binding antibodies of the invention do not activate Tcells in the absence of target cells, since a monovalent CD3 stimulus isnot sufficient to initiate effective T cell activation. However, in somecases, there may be some deviation from this assumed behavior. WhenUCHT1 was used here as a CD3 antibody within a bispecific Fabscconstruct, some T cell activation was noted in the absence of PSMAexpressing target cells. These findings were considerably morepronounced when the experiments were performed in the presence ofstimulating bystander cells, such as SKW6.4 lymphoma cells. In contrastthereto, the inventors have surprisingly also found here that antibodiescontaining OKT3 and -surprisingly - also the IgGsc antibody comprisingUCHT1 induce markedly attenuated off-target T cell activation. Theunexpectedly low off-target activation by the IgGsc antibody comprisingUCHT1 is at least in part explained by avidity measurements using CD3expressing Jurkat cells. These experiments demonstrate that UCHT1 losesand OKT3 gains avidity if expressed in the IgGsc (rather than the Fabsc)format (cf. FIG. 8 ). When tested in a long term cytotoxic assay(Xelligence) against PSMA expressing 22RV1 cells the inventors of thepresent invention have surprisingly found following ranking forcytolytic activity: IgGsc(UCHT1) ~ Fabsc(UCHT1) > Fabsc(OKT3) >IgGsc(OKT3). Thus, within the IgGsc-format, UCHT1 may be the preferredCD3 antibody (favorable off-target activation and cytolytic activity),whereas within the Fabsc-format OKT3 may be preferred due to theundesirably high off-target activation by the UCHT1 containing Fabsc(cf. FIG. 9 ).

Regardless of the particular CD3 antibody used, clustering of bispecificantibodies on the surface of a T cell, induced by the interactionbetween targeting single chain fragments, may also lead to off-target Tcell activation in the absence of a target cell. To prevent thisphenomenon it may be desirable to use a Fab- or Fab₂-moiety, such as theone contained in the Fabsc- or IgGsc-format, respectively, rather than asingle chain antibody as targeting part within a bispecific construct.Multimerization and aggregation of bispecific antibodies expressed insuch formats is markedly reduced compared to that observed withbispecific single chain antibodies. FIGS. 4A and 4B show thataggregation of a bispecific single chain format with FLT3xCD3specificity is less pronounced than that of an otherwise identicalantibody in the bispecific single chain (bssc) or BiTE-format, asdetermined by size exclusion chromatography. FIGS. 4C-F show that,likewise, the tendency of the four PSMAxCD3 antibodies in the Fabsc aswell as in the IgGsc format, to form multimers or aggregates is stronglyreduced. Notably, the inventors of the present invention havesurprisingly found that the formation of multimers is even considerablyless pronounced in the two constructs containing the UCHT1 antibodycompared to the constructs comprising the OKT3 antibody. In any case, itis believed that aggregation, if it occurs, is due to the CD3 effectorpart expressed as a single chain. It is believed and found here that thephysiological Fab- and Fab₂ moieties at the N-terminal targeting part donot aggregate and thus it is believed that single chain clustering ofthe N-terminal targeting part, possibly resulting in T cell exhaustionin vivo, cannot occur in a respective antibody molecule of theinvention.

Further, the inventors here foresee that the serum half-life of anantibody molecule is largely determined by the interaction of CH3domains with the FcRn receptor. Since most bispecific antibodies arelacking this domain, their serum half-life may be rather short (severalhours at best). In contrast, whole IgG molecules usually have a serumhalf-life of several days. Although IgG-based bispecific formats havebeen available for several years, they have been rarely used forconstruction of CD3-stimulating antibodies because it was thought that abivalent CD3 stimulus may lead to off-target T cell activation. However,the inventors of the present invention have surprisingly found that thecontrary is true for two different formats containing the UCHT1antibody: off-target T cell activation by the Fabsc format is markedlyhigher than that by the antibody in the IgGsc-format. Without wishing tobe bound to theory, it is believed that this is because the avidity ofthe CD3 moiety in the latter format is impaired (cf. again FIG. 8 ).This means, in the case of an antibody molecule comprising the variableregion of UCHT1, the IgGsc- format surprisingly provides not only amarkedly improved serum half-life but also reduced off-target T cellactivation. Hence, if a prolonged serum half-life is desired, e.g. toavoid long term continuous infusion, a 10B3xUCHT1 bispecific antibody inthe IgGsc format may be preferred, wherein the Fab moiety comprises theantigen binding site of an antibody derived from 10B3 and wherein thescFv moiety comprises the antigen binding site of an antibody derivedfrom UCHT1. If a prolonged serum half-life is not desired, 10B3xOKT3bispecific antibody in the Fabsc format may be preferred, wherein theFab moiety comprises the antigen binding site of an antibody derivedfrom 10B3 and wherein the scFv moiety comprises the antigen binding siteof an antibody derived from OKT3.

Therefore, in other words, the present invention in an alternativeaspect further pertains to a tetravalent and homodimeric bispecificantibody molecule (a bispecific antibody in the herein described IgGscformat) comprising in each monomer: (i) an N-terminal Fab fragmentcomprising a variable region comprising a heavy chain variable domainand a light chain variable domain, wherein said variable regioncomprises a first binding site capable of binding to an antigen; (ii) aC-terminal scFv fragment, comprising a heavy chain variable region and alight chain variable region of humanized UCHT1, and wherein (i) and (ii)are connected by a CH2 and CH3 domain

In some embodiments the tetravalent bispecific antibody molecule of theinvention is preferred, wherein at least one amino acid residue of theCH2 domain that is able to mediate binding to Fc receptors is lacking ormutated (see also herein elsewhere).

In some embodiments the tetravalent bispecific antibody molecule of theinvention is preferred, wherein the Fab fragment is not a Fab fragmentof a non-humanized, chimerized or humanized 10B3 or J591 antibody,preferably wherein the first binding site is not capable of binding toPSMA. Therefore, in some preferred embodiments the first binding siteprovided by the N-terminal Fab fragment is not specific for PSMA, butfor a non-PSMA antigen, preferably a tumor associated antigen exceptPSMA. The term “tumor-associated antigen” relates to proteins that areunder normal conditions, i.e. in a healthy subject, specificallyexpressed in a limited number of organs and/or tissues or in specificdevelopmental stages, for example, the tumor-associated antigen may beunder normal conditions specifically expressed in stomach tissue,preferably in the gastric mucosa, in reproductive organs, e.g., intestis, in trophoblastic tissue, e.g., in placenta, or in germ linecells, and are expressed or aberrantly expressed in one or more tumor orcancer tissues. In this context, “a limited number” preferably means notmore than 3, more preferably not more than 2 or 1. The tumor-associatedantigens in the context of the present invention include, for example,differentiation antigens, preferably cell type specific differentiationantigens, i.e., proteins that are under normal conditions specificallyexpressed in a certain cell type at a certain differentiation stage,cancer/testis antigens, i.e., proteins that are under normal conditionsspecifically expressed in testis and sometimes in placenta, and germline specific antigens. In the context of the present invention, thetumor-associated antigen is preferably not or only rarel expressed innormal tissues or is mutated in tumor cells. Preferably, thetumor-associated antigen or the aberrant expression of thetumor-associated antigen identifies cancer cells. In the context of thepresent invention, the tumor-associated antigen that is expressed by acancer cell in a subject, e.g., a patient suffering from a cancerdisease is preferably a self-protein in said subject. In preferredembodiments, the tumor-associated antigen in the context of the presentinvention is expressed under normal conditions specifically in a tissueor organ that is non-essential, i.e., tissues or organs which whendamaged by the immune system do not lead to death of the subject, or inorgans or structures of the body which are not or only hardly accessibleby the immune system. Preferably, a tumor-associated antigen ispresented in the context of MHC molecules by a cancer cell in which itis expressed.

Examples for TAA that may be useful in the present invention and thatare not PSMA - according to preferred embodiments of the presentaspect - are p53, ART-4, BAGE, beta-catenin/m, Bcr-abL CAMEL, CAP-1,CASP-8, CDC27/m, CDK4/m, CEA, CLAUD1N-12, c-MYC, CT, Cyp-B, DAM, ELF2M,ETV6-AML1, G250, GAGE, GnT-V, Gap 100, HAGE, HER-2/neu, HPV-E7, HPV-E6,HAST-2, hTERT (or hTRT), LAGE, LDLR/FUT, MAGE-A, preferably MAGE-A1,MAGE-A2, MAGE- A3, MAGE-A4, MAGE- A5, MAGE-A6, MAGE-A7, MAGE-A8,MAGE-A9, MAGE- A 10, MAGE-A11 , or MAGE- A12, MAGE-B, MAGE-C, MART- 1/Melan- A, MC1R, Myosin/m, MUCl”, MUM-1 , -2, -3, NA88-A, NF1, NY-ESO-1,NY-BR-1 , pl90 minor BCR-abL, Pml /RARa, PRAME, proteinase 3, PSA, RAGE,RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX,SURVIVIN, TEL/AML1, TPl/m, TRP-1, TRP-2, TRP-2/INT2, TPTE and WT.

In accordance with the present aspect of the invention a humanized UCHT1is preferably a scFv fragment comprising the light chain variable regionsequence and heavy chain variable region sequence as shown in SEQ ID NO:11 starting with the amino acid sequence DIQMT (SEQ ID NO: 17)... andending with... VTVSS (SEQ IDS NO: 18) (as indicated in FIG. 11 ).Preferably the light chain variable region sequence and heavy chainvariable region sequence in the scFv fragment are connected via a linkeras depicted in FIG. 11 (“linker region”).

It is noted in this context that it is within the scope of the inventionthat an antibody molecule may comprise one or more mutated amino acidresidues. The terms “mutated”, “mutant” and “mutation” in reference to anucleic acid or a polypeptide refers to the exchange, deletion, orinsertion of one or more nucleotides or amino acids, respectively,compared to the “naturally” occurring nucleic acid or polypeptide, i.e.to a reference sequence that can be taken to define the wild-type. Forexample, the variable domains of the antibody molecule 10B3 as obtainedby immunization and as described herein may be taken as a wild-typesequence.

It is understood in this regard that the term “position”, when used inaccordance with the present invention, means the position of an aminoacid within an amino acid sequence depicted herein. This position may beindicated relative to a resembling native sequence, e.g. a sequence of anaturally occurring IgG domain or chain. The term “corresponding” asused herein also includes that a position is not necessarily, or notonly, determined by the number of the preceding nucleotides/amino acids.Thus, the position of a given amino acid in accordance with the presentinvention which may be substituted may vary due to deletion or additionof amino acids elsewhere in the antibody chain.

Thus, under a “corresponding position” in accordance with the presentinvention it is to be understood that amino acids may differ in theindicated number but may still have similar neighbouring amino acids.Said amino acids which may be exchanged, deleted or added are alsoencompassed by the term “corresponding position”. In order to determinewhether an amino acid residue in a given amino acid sequence correspondsto a certain position in the amino acid sequence of a naturallyoccurring immunoglobulin domain or chain, the skilled person can usemeans and methods well-known in the art, e.g., alignments, eithermanually or by using computer programs such as BLAST2.0, which standsfor Basic Local Alignment Search Tool or ClustalW or any other suitableprogram which is suitable to generate sequence alignments.

In some embodiments a substitution (or replacement) is a conservativesubstitution. Conservative substitutions are generally the followingsubstitutions, listed according to the amino acid to be mutated, eachfollowed by one or more replacement(s) that can be taken to beconservative: Ala → Gly, Ser, Val; Arg → Lys; Asn → Gln, His; Asp → Glu;Cys → Ser; Gln → Asn; Glu → Asp; Gly → Ala; His → Arg, Asn, Gln; Ile →Leu, Val; Leu → Ile, Val; Lys → Arg, Gln, Glu; Met → Leu, Tyr, Ile; Phe→ Met, Leu, Tyr; Ser → Thr; Thr → Ser; Trp → Tyr; Tyr → Trp, Phe; Val →Ile, Leu. Other substitutions are also permissible and can be determinedempirically or in accord with other known conservative ornon-conservative substitutions. As a further orientation, the followingeight groups each contain amino acids that can typically be taken todefine conservative substitutions for one another:

-   Alanine (Ala), Glycine (Gly);-   Aspartic acid (Asp), Glutamic acid (Glu);-   Asparagine (Asn), Glutamine (Gln);-   Arginine (Arg), Lysine (Lys);-   Isoleucine (Ile), Leucine (Leu), Methionine (Met), Valine (Val);-   Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp);-   Serine (Ser), Threonine (Thr); and-   Cysteine (Cys), Methionine (Met)

If such substitutions result in a change in biological activity, thenmore substantial changes, such as the following, or as further describedbelow in reference to amino acid classes, may be introduced and theproducts screened for a desired characteristic. Examples of such moresubstantial changes are: Ala → Leu, Ile; Arg → Gln; Asn → Asp, Lys, Arg,His; Asp → Asn; Cys → Ala; Gln → Glu; Glu → Gln; His → Lys; Ile → Met,Ala, Phe; Leu → Ala, Met, Norleucine; Lys → Asn; Met → Phe; Phe → Val,Ile, Ala; Trp → Phe; Tyr → Thr, Ser; Val → Met, Phe, Ala.

In some embodiments an antibody molecule according to the inventionincludes one or more amino acid residues, including two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, seventeen or eighteen amino acid residues, that aremutated to prevent dimerization via cysteine residues or to modulateFc-function (see above). In some of these embodiments one or more aminoacid residue(s) of the CH2 domain and/or of the hinge region that isable to mediate binding to Fc receptors are mutated. If present, the oneor more amino acid residue(s) able to mediate binding to Fc receptorsmay be an amino acid residue that is able to activate antibody dependentcellular cytotoxicity (ADCC) or complement-mediated cytotoxicity (CDC).In some embodiments a respective amino acid residue capable of mediatingbinding to Fc receptors is substituted by another amino acid, generallywhen comparing the sequence to the sequence of a corresponding naturallyoccurring domain in an immunoglobulin, such as an IgG In someembodiments such an amino acid residue capable of mediating binding toFc receptors is deleted, generally relative to the sequence of acorresponding naturally occurring domain in an immunoglobulin, such asan IgG

In some embodiments the one or more mutated, e.g. substituted ordeleted, amino acid residues is/are an amino acid located at one of thepositions 226, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,265, 297, 327, and 330. Again, the numbering of amino acids usedcorresponds to the sequence positions according to the Kabat numbering[EU-Index]. A corresponding deletion of an amino acid may for example bea deletion of amino acid 228, generally a proline in IgG, a deletion ofamino acid 229, generally a cysteine in IgG, a deletion of amino acid230, generally a proline in IgG, a deletion of amino acid 231, generallyan alanine in IgG, a deletion of amino acid 232, generally a proline inIgG, a deletion of amino acid 233, generally a glutamic acid in IgG, adeletion of amino acid 234, generally a leucine in IgG, a deletion ofamino acid 235, generally a leucine in IgG, a deletion of amino acid236, generally a glycine in IgG, a deletion of amino acid 237, generallya glycine in IgG, a deletion of amino acid 238, generally a proline inIgG and a deletion of amino acid 265, generally an aspartic acid in IgGA corresponding substitution of an amino acid may for example be asubstitution of amino acid 226, generally a cysteine in IgG, asubstitution of amino acid 228, generally a proline in IgG, asubstitution of amino acid 229, generally a cysteine in IgG, asubstitution of amino acid 230, generally a proline in IgG, asubstitution of amino acid 231, generally an alanine in IgG, asubstitution of amino acid 232, generally a proline in IgG, asubstitution of amino acid 233, generally a glutamic acid in IgG, asubstitution of amino acid 234, generally a leucine in IgG, asubstitution of amino acid 235, generally a leucine in IgG, asubstitution of amino acid 265, generally an aspartic acid in IgG, asubstitution of amino acid 297, generally an asparagine in IgG, asubstitution of amino acid 327, generally an alanine in IgG, and asubstitution of amino acid 330, generally an alanine in IgG A respectivesubstitution may be one of substitution Cys226→Ser, substitutionCys229→Ser, substitution Glu233→Pro, substitution Leu234→Val,substitution Leu235→Ala, substitution Asp265→Gly, substitutionAsn297→Gln, substitution Ala327→Gln, substitution Ala327→Gly, andsubstitution Ala330→Ser. As can be taken from the above, in someembodiments one or two of the cysteine residues at positions 226 and 229in the hinge region are being substituted for another amino acid, forinstance substituted for a serine residue. Thereby the formation of adisulphide bond with another main chain can be prevented. Further, andas also explained below, deleting and/ or substituting (mutating)selected amino acid residues in the CH2 domain that is able to mediatebinding to Fc-receptors can cause an antibody molecule of the inventionto have less or no activity in terms of antibody-dependent cell-mediatedcytotoxicity and fixation of complement.

Another type of amino acid variant of an antibody alters the originalglycosylation pattern (if any) of the antibody molecule. By altering ismeant deleting one or more carbohydrate moieties found in the antibody,and/or adding one or more glycosylation sites that are not present inthe antibody. Glycosylation of antibodies is typically either N-linkedor O-linked. N-linked refers to the attachment of the carbohydratemoiety to the side chain of an asparagine residue. The tripeptidesequences asparagine-X-serine and asparagine-X-threonine, where X is anyamino acid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. Addition of glycosylation sites to theantibody is conveniently accomplished by altering the amino acidsequence such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the original antibody(for O-linked glycosylation sites).

In the context of the present invention, in some embodiments the portionof the main chain of the antibody molecule of the invention, whichrepresents the Fc region of an immunoglobulin, is typically inert, or atleast essentially of low influence, with regard to binding to Fcreceptors. As said, this is achieved by deleting and/ or substituting(mutating) at least one of selected amino acid residues in the CH2domain that are able to mediate binding to an Fc-receptor. Suchmolecules are also referred to herein as “Fc-attenuated” antibodymolecules or “Fc^(ko)” antibody molecules. The portion of an antibodychain according to the invention that can be taken to represent aportion of an Fc fragment, i.e. the CH2 domain, and, where present, theCH3 domain, thus might define a “scaffold” without providing aparticular biological function such as an effector function, forexample. However, it has been found in the present invention, that thisscaffold may provide significant advantages in terms of purification,production efficiency and/or stability of the antibody molecules of theinvention compared to known antibody molecules.

In some embodiments the recognition, and accordingly binding, of thisFc-corresponding portion to a given Fc receptor is of about 2-fold,about 5-fold, about 8-fold, about 10-fold, about 12-fold, about 15-fold,about 20-fold or lower than the Fc region of a naturally occurringimmunoglobulin. In some embodiments this Fc-corresponding portion isentirely void of its ability of binding to Fc receptors. The binding ofan antibody to Fc receptors, including determining a dissociationconstant, can easily be determined by the skilled artisan using standardtechniques such as surface plasmon resonance, e.g. using a Biacore™measurement. Any other method of measuring biomolecular binding maylikewise be used, which may for instance rely on spectroscopical,photochemical, photometric or radiological means. Examples for thecorresponding detection methods are fluorescence correlationspectroscopy, photochemical cross-linking and the use of photoactive orradioactive labels respectively. Some of these methods may includeadditional separation techniques such as electrophoresis or HPLC.

Where required, a substitution or deletion of amino acid residues, asexplained above, may be carried out to this effect. Suitable mutationscan be taken from Armour et al. (Eur. J. Immunol. [1999] 29, 2613-2624),for example. Further suitable positions for mutations to a sequence ofan antibody chain can be taken from the crystal structure data publishedon the complex between FcγRIII and the human IgG1 Fc fragment(Sondermann et al., Nature [2000] 406, 267-273). In addition tomeasuring the binding affinity as described above in order to assess thelevel of “Fc attenuation” or loss of binding affinity, it is alsopossible to functionally assess the (lack of the) ability to mediatebinding to an Fc-receptor. In the case of antibody molecules which bindCD3 as one target, it is for example possible to assess the bindingthrough the mitogenity of such CD3 binding antibody molecules on cells.The mitogenity is mediated by binding of CD3 antibodies to theFc-receptors on accessory cells, such as monocytes. If an antibodymolecule of the invention that has one binding site for CD3 does notshow any mitogenic effect whereas the parent monoclonal anti-CD3antibody that has a functional Fc part induces strong mitosis in Tcells, it is clear that, due to the lack of mitosis, the antibodymolecule of the invention lacks the ability for Fc binding and can thusbe considered as a “Fc knock-out” molecule. Illustrative examples of amethod of assessing anti-CD3 mediated mitogenity have been described byDavis, Vida & Lipsky (J.Immunol (1986) 137, 3758), and by Ceuppens, JL,& van Vaeck, F, (see J.Immunol. (1987) 139, 4067, or Cell. Immunol.(1989) 118, 136). Further illustrative suitable examples of an assay forassessing mitogenity of an antibody have been described byRosenthal-Allieri et al. (Rosenthal-Allieri MA, Ticcioni M, Deckert M,Breittmeyer JP, Rochet N, Rouleaux M, and Senik A, Bernerd A, CellImmunol. 1995 163(1):88-95) and Grosse-Hovest et al. (Grosse-Hovest L,Hartlapp I, Marwan W, Brem G, Rammensee H-G, and Jung G, Eur J Immunol.[2003] May;33(5):1334-1340). In addition, the lack of Fc binding can beassessed by the ability of an antibody molecule of the invention tomediate one or more of the well-known effector functions of the Fc part.

As noted above, substitutions or deletions of cysteine residues may becarried out in order to introduce or to remove one or more disulfidebonds, including introducing or removing a potential or a previouslyexisting disulfide bond. Thereby linkage between a main chain and achain of lower weight/shorter length of an antibody molecule accordingto the invention may be controlled including established, strengthenedor abolished. By introducing or removing one or more cysteine residues adisulfide bridge may be introduced or removed. As an illustrativeexample, a tetrameric antibody molecule according to the inventiongenerally has one or more disulfide bonds that link two dimeric antibodymolecules. One such disulfide bond is typically defined by a cysteine inthe main chain of a first dimeric antibody molecule and a cysteine inthe hinge region of a second dimeric antibody molecule. In this regard,in some embodiments an antibody according to the invention may includean amino acid substitution of a native cysteine residue at positions 226and/or 229, relative to the sequence of a human IgG immunoglobulinaccording to the Kabat numbering [EU-Index], by another amino acidresidue.

Substitutions or deletions of amino acid residues such as arginine,asparagine, serine, threonine or tyrosine residues may also be carriedout to modify the glycosylation pattern of an antibody. As anillustrative example, an IgG molecule has a single N-linked biantennarycarbohydrate at Asn297 of the CH2 domain. For IgG from either serum orproduced ex vivo in hybridomas or engineered cells, the IgG areheterogeneous with respect to the Asn297 linked carbohydrate. For humanIgG, the core oligosaccharide typically consists of GlcNAc₂Man₃GlcNAc,with differing numbers of outer residues.

As indicated, besides binding of antigens/epitopes, an immunoglobulin isknown to have further “effector functions”, biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an immunoglobulin, and vary with theimmunoglobulin isotype. Examples of antibody effector functions include:Clq binding and complement dependent cytotoxicity (CDC); Fc receptorbinding; antibody-dependent cell-mediated cytotoxicity (ADCC);phagocytosis; down regulation of cell surface receptors (e.g., B cellreceptors); and B cell activation. Exerting effector functions of anantibody generally involves recruiting effector cells. Severalimmunoglobulin effector functions are mediated by Fc receptors (FcRs),which bind the Fc region of an antibody. FcRs are defined by theirspecificity for immunoglobulin isotypes; Fc receptors for IgG antibodiesare referred to as FcγR, for IgE as FcεR, for IgA as FcαR and so on. Anyof these effector functions (or the loss of such effector functions)such a CDC or ADCC can be used in order to evaluate whether an antibodymolecule of the invention lacks the ability of Fc binding.

In this context, it is noted that the term “Fc receptor” or “FcR”defines a receptor, generally a protein that is capable of binding tothe Fc region of an antibody. Fc receptors are found on the surface ofcertain cells of the immune system of an organism, for example naturalkiller cells, macrophages, neutrophils, and mast cells. In vivo Fcreceptors bind to immunoglobulins that are immobilized on infected cellsor present on invading pathogens. Their activity stimulates phagocyticor cytotoxic cells to destroy microbes, or infected cells byantibody-mediated phagocytosis or antibody-dependent cell-mediatedcytotoxicity. Some viruses such as flaviviruses use Fc receptors to helpthem infect cells, by a mechanism known as antibody-dependentenhancement of infection. FcRs have been reviewed in Ravetch and Kinet,Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (Clq) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202: 163 (1997) may be performed.

The term “complement system” is used in the art to refer a number ofsmall proteins - called complement factors - found in blood, generallycirculating as inactive precursors (pro-proteins). The term refers tothe ability of this inalterable and not adaptable system to “complement”the capability of antibodies and phagocytic cells to clear pathogenssuch as bacteria, as well as antigen-antibody complexes, from anorganism. An example of complement factors is the complex C1, whichincludes C1q and two serine protases, C1r and C1s. The complex C1 is acomponent of the CDC pathway. C1q is a hexavalent molecule with amolecular weight of approximately 460,000 and a structure likened to abouquet of tulips in which six collagenous “stalks” are connected to sixglobular head regions. To activate the complement cascade, C1q has tobind to at least two molecules of IgG1, IgG2 or IgG3.

“Antibody-dependent cellular cytotoxicity” or ADCC refers to a form ofcytotoxicity in which immunoglobulin molecules, bound onto Fc receptors(FcRs), present on certain cytotoxic cells - such as natural killer (NK)cells, neutrophils and macrophages -enable these cytotoxic effectorcells to bind specifically to an antigen-bearing target cell and tosubsequently kill the target cell with cytotoxins. The antibodies “arm”the cytotoxic cells and are required for killing of the target cell bythis mechanism. The primary cells for mediating ADCC, NK cells, expressFcγRIII only, whereas monocytes express FcγRI, FcγRlI and FcγRIII. FcRexpression on hematopoietic cells is summarized in Table 3 on page 464of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991). To assessADCC activity of a molecule of interest, an in vitro ADCC assay, such asdescribed in U.S. Pat. Nos. 5,500,362 or 5,821,337 may be carried out.Useful effector cells for such assays include, but are not limited to,peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.In some embodiments ADCC activity of the molecule of interest may beassessed in vivo, e.g., in an animal model such as disclosed in Clyneset al., PNAS USA 95: 652-656 (1998).

An antibody molecule of the invention may be produced using any knownand well-established expression system and recombinant cell culturingtechnology, for example, by expression in bacterial hosts (prokaryoticsystems), or eukaryotic systems such as yeasts, fungi, insect cells ormammalian cells. For example, an antibody molecule of the invention whenbeing used in the “IgGsc” format, the antibody molecule can (of course)be produced as described by Coloma and Morrison (Nat Biotechnol15:159-63, 1997) or as described in the Example Section of the presentapplication. Likewise, an antibody molecule of the invention employed inthe “Fabsc” format can be produced as described in International patentapplication WO 2013/092001 or as described here in the Example Section.An antibody molecule of the present invention may also be produced intransgenic organisms such as a goat, a plant or a XENOMOUSE transgenicmouse, an engineered mouse strain that has large fragments of the humanimmunoglobulin loci and is deficient in mouse antibody production. Anantibody may also be produced by chemical synthesis.

For production of a recombinant antibody molecule of the invention,typically a polynucleotide encoding the antibody is isolated andinserted into a replicable vector such as a plasmid for further cloning(amplification) or expression. An illustrative example of a suitableexpression system is a glutamate synthetase system (such as sold byLonza Biologics), with the host cell being for instance CHO or NS0. Apolynucleotide encoding the antibody is readily isolated and sequencedusing conventional procedures. Vectors that may be used include plasmid,virus, phage, transposons, minichromsomes of which plasmids are atypical embodiment. Generally such vectors further include a signalsequence, origin of replication, one or more marker genes, an enhancerelement, a promoter and transcription termination sequences operablylinked to the light and/or heavy chain polynucleotide so as tofacilitate expression. Polynucleotides encoding the light and heavychains may be inserted into separate vectors and transfected into thesame host cell or, if desired both the heavy chain and light chain canbe inserted into the same vector for transfection into the host cell.Both chains can, for example, be arranged, under the control of adicistronic operon and expressed to result in the functional andcorrectly folded antibody molecule as described in Skerra, A. (1994) Useof the tetracycline promoter for the tightly regulated production of amurine antibody fragment in Escherichia coli, Gene 151, 131-135, orSkerra, A. (1994) A general vector, pASK84, for cloning, bacterialproduction, and single-step purification of antibody Fab fragments, Gene141, 79-8. Thus according to one aspect of the present invention thereis provided a process of constructing a vector encoding the light and/orheavy chains of an antibody or antigen binding fragment thereof of theinvention, which method includes inserting into a vector, apolynucleotide encoding either a light chain and/or heavy chain of anantibody molecule of the invention.

When using recombinant techniques, the antibody molecule can be producedintracellularly, in the periplasmic space, or directly secreted into themedium (cf. also Skerra 1994, supra). If the antibody is producedintracellularly, as a first step, the particulate debris, either hostcells or lysed fragments, are removed, for example, by centrifugation orultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992)describe a procedure for isolating antibodies which are secreted to theperiplasmic space of E coli. The antibody can also be produced in anyoxidizing environment. Such an oxidizing environment may be provided bythe periplasm of Gram-negative bacteria such as E. coli, in theextracellular milieu of Gram-positive bacteria or in the lumen of theendoplasmatic reticulum of eukaryotic cells (including animal cells suchas insect or mammalian cells) and usually favors the formation ofstructural disulfide bonds. It is, however, also possible to produce anantibody molecule of the invention in the cytosol of a host cell such asE. coli. In this case, the polypeptide can either be directly obtainedin a soluble and folded state or recovered in form of inclusion bodies,followed by renaturation in vitro. A further option is the use ofspecific host strains having an oxidizing intracellular milieu, whichmay thus allow the formation of disulfide bonds in the cytosol (VenturiM, Seifert C, Hunte C. (2002) “High level production of functionalantibody Fab fragments in an oxidizing bacterial cytoplasm.” J. Mol.Biol. 315, 1-8).

The antibody molecule produced by the cells can be purified using anyconventional purification technology, for example, hydroxylapatitechromatography, gel electrophoresis, dialysis, and affinitychromatography, with affinity chromatography being one preferredpurification technique. Antibody molecules may be purified via affinitypurification with proteins/ligands that specifically and reversibly bindconstant domains such as the CH1 or the CL domains. Examples of suchproteins are immunoglobulin-binding bacterial proteins such as ProteinA, Protein G, Protein A/G or Protein L, wherein Protein L binding isrestricted to antibody molecules that contain kappa light chains. Analternative method for purification of antibodies with κ-light chains isthe use of bead coupled anti kappa antibodies (KappaSelect). Thesuitability of protein A as an affinity ligand depends on the speciesand isotype of any immunoglobulin Fc domain that is present in theantibody. Protein A can be used to purify antibodies (Lindmark et al.,J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for allmouse isotypes and for human gamma3 (Guss et al., EMBO J. 5: 15671575(1986)). The choice of the purification method that is used for aparticular antibody molecule of the invention is within the knowledge ofthe person of average skill in the art.

It is also possible to equip one of the chains of the antibody moleculeof the invention with one or more affinity tags. Affinity tags such asthe Strep-tag® or Strep-tag® II (Schmidt, T.GM. et al. (1996) J. Mol.Biol. 255, 753-766), the myc-tag, the FLAG™-tag, the His6-tag or theHA-tag allow easy detection and also simple purification of therecombinant antibody molecule.

Turning now to nucleic acids of the invention, a nucleic acid moleculeencoding one or more chains of an antibody according to the inventionmay be any nucleic acid in any possible configuration, such as singlestranded, double stranded or a combination thereof. Nucleic acidsinclude for instance DNA molecules, RNA molecules, analogues of the DNAor RNA generated using nucleotide analogues or using nucleic acidchemistry, locked nucleic acid molecules (LNA), and protein nucleicacids molecules (PNA). DNA or RNA may be of genomic or synthetic originand may be single or double stranded. Such nucleic acid can be e.g.mRNA, cRNA, synthetic RNA, genomic DNA, cDNA synthetic DNA, a copolymerof DNA and RNA, oligonucleotides, etc. A respective nucleic acid mayfurthermore contain non-natural nucleotide analogues and/or be linked toan affinity tag or a label.

In some embodiments a nucleic acid sequence encoding a chain, such as amain chain and/or a smaller chain of an antibody according to theinvention is included in a vector such as a plasmid. Where asubstitution or deletion is to be included in an antibody chain, whencompared to a naturally occurring domain or region of an antibody, thecoding sequence of the respective native domain/region, e.g. included inthe sequence of an immunoglobulin, can be used as a starting point forthe mutagenesis. For the mutagenesis of selected amino acid positions,the person skilled in the art has at his disposal the variousestablished standard methods for site-directed mutagenesis. A commonlyused technique is the introduction of mutations by means of PCR(polymerase chain reaction) using mixtures of syntheticoligonucleotides, which bear a degenerate base composition at thedesired sequence positions. For example, use of the codon NNK or NNS(wherein N = adenine, guanine or cytosine or thymine; K = guanine orthymine; S = adenine or cytosine) allows incorporation of all 20 aminoacids plus the amber stop codon during mutagenesis, whereas the codonVVS limits the number of possibly incorporated amino acids to 12, sinceit excludes the amino acids Cys, Ile, Leu, Met, Phe, Trp, Tyr, Val frombeing incorporated into the selected position of the polypeptidesequence; use of the codon NMS (wherein M = adenine or cytosine), forexample, restricts the number of possible amino acids to 11 at aselected sequence position since it excludes the amino acids Arg, Cys,Gly, Ile, Leu, Met, Phe, Trp, Val from being incorporated at a selectedsequence position. In this respect it is noted that codons for otheramino acids (than the regular 20 naturally occurring amino acids) suchas selenocystein or pyrrolysine can also be incorporated into a nucleicacid of an antibody molecule. It is also possible, as described by Wang,L., et al. (2001) Science 292, 498-500, or Wang, L., and Schultz, P.G.(2002) Chem. Comm. 1, 1-11, to use “artificial” codons such as UAG whichare usually recognized as stop codons in order to insert other unusualamino acids, for example o-methyl-L-tyrosine or p-aminophenylalanine.

The use of nucleotide building blocks with reduced base pairspecificity, as for example inosine, 8-oxo-2′deoxyguanosine or6(2-deoxy-(β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimin-do-1,2-oxazine-7-one(Zaccolo et al. (1996) J. Mol. Biol. 255, 589-603), is another optionfor the introduction of mutations into a chosen sequence segment. Afurther possibility is the so-called triplet-mutagenesis. This methoduses mixtures of different nucleotide triplets, each of which codes forone amino acid, for incorporation into the coding sequence (Virnekäs B,et al., 1994 Nucleic Acids Res 22, 5600-5607).

A nucleic acid molecule encoding a chain, such as a main chain and/or asmaller chain of an antibody according to the invention can be expressedusing any suitable expression system, for example in a suitable hostcell or in a cell-free system. The obtained antibody molecule may beenriched by means of selection and/ or isolation.

The invention also provides a pharmaceutical composition that includesan antibody molecule of the invention and, optionally a pharmaceuticallyacceptable excipient.

The antibody molecule according to the invention can be administered viaany parenteral or non-parenteral (enteral) route that is therapeuticallyeffective for proteinaceous drugs. Parenteral application methodsinclude, for example, intracutaneous, subcutaneous, intramuscular,intratracheal, intranasal, intravitreal or intravenous injection andinfusion techniques, e.g. in the form of injection solutions, infusionsolutions or tinctures, as well as aerosol installation and inhalation,e.g. in the form of aerosol mixtures, sprays or powders. An overviewabout pulmonary drug delivery, i.e. either via inhalation of aerosols(which can also be used in intranasal administration) or intrachealinstillation is given by J.S. Patton et al. The lungs as a portal ofentry for systemic drug delivery. Proc. Amer. Thoracic Soc. 2004 Vol. 1pages 338-344, for example). Non-parenteral delivery modes are, forinstance, orally, e.g. in the form of pills, tablets, capsules,solutions or suspensions, or rectally, e.g. in the form ofsuppositories. Antibody molecules of the invention can be administeredsystemically or topically in formulations containing conventionalnon-toxic pharmaceutically acceptable excipients or carriers, additivesand vehicles as desired.

In one embodiment of the present invention the pharmaceutical isadministered parenterally to a mammal, and in particular to humans.Corresponding administration methods include, but are not limited to,for example, intracutaneous, subcutaneous, intramuscular, intratrachealor intravenous injection and infusion techniques, e.g. in the form ofinjection solutions, infusion solutions or tinctures as well as aerosolinstallation and inhalation, e.g. in the form of aerosol mixtures,sprays or powders. A combination of intravenous and subcutaneousinfusion and /or injection might be most convenient in case of compoundswith a relatively short serum half-life. The pharmaceutical compositionmay be an aqueous solution, an oil-in water emulsion or a water-in-oilemulsion.

In this regard it is noted that transdermal delivery technologies, e.g.iontophoresis, sonophoresis or microneedle-enhanced delivery, asdescribed in Meidan VM and Michniak BB 2004 Am. J. Ther. 11(4): 312-316,can also be used for transdermal delivery of an antibody moleculedescribed herein. Non-parenteral delivery modes are, for instance, oral,e.g. in the form of pills, tablets, capsules, solutions or suspensions,or rectal administration, e.g. in the form of suppositories. Theantibody molecules of the invention can be administered systemically ortopically in formulations containing a variety of conventional non-toxicpharmaceutically acceptable excipients or carriers, additives, andvehicles.

The dosage of the antibody molecule applied may vary within wide limitsto achieve the desired preventive effect or therapeutic response. Itwill, for instance, depend on the affinity of the antibody molecule fora chosen target as well as on the half-life of the complex between theantibody molecule and the ligand in vivo. Further, the optimal dosagewill depend on the biodistribution of the antibody molecule or aconjugate thereof, the mode of administration, the severity of thedisease/disorder being treated as well as the medical condition of thepatient. For example, when used in an ointment for topical applications,a high concentration of the antibody molecule can be used. However, ifwanted, the antibody molecule may also be given in a sustained releaseformulation, for example liposomal dispersions or hydrogel-based polymermicrospheres, like PolyActiveTM or OctoDEXTM (cf. Bos et al., BusinessBriefing: Pharmatech 2003: 1-6). Other sustained release formulationsavailable are for example PLGA based polymers (PR pharmaceuticals),PLA-PEG based hydrogels (Medincell) and PEA based polymers (Medivas).

Accordingly, the antibody molecules of the present invention can beformulated into compositions using pharmaceutically acceptableingredients as well as established methods of preparation (Gennaro, A.L.and Gennaro, A.R. (2000) Remington: The Science and Practice ofPharmacy, 20th Ed., Lippincott Williams & Wilkins, Philadelphia, PA). Toprepare the pharmaceutical compositions, pharmaceutically inertinorganic or organic excipients can be used. To prepare e.g. pills,powders, gelatin capsules or suppositories, for example, lactose, talc,stearic acid and its salts, fats, waxes, solid or liquid polyols,natural and hardened oils can be used. Suitable excipients for theproduction of solutions, suspensions, emulsions, aerosol mixtures orpowders for reconstitution into solutions or aerosol mixtures prior touse include water, alcohols, glycerol, polyols, and suitable mixturesthereof as well as vegetable oils.

The pharmaceutical composition may also contain additives, such as, forexample, fillers, binders, wetting agents, glidants, stabilizers,preservatives, emulsifiers, and furthermore solvents or solubilizers oragents for achieving a depot effect. The latter is that fusion proteinsmay be incorporated into slow or sustained release or targeted deliverysystems, such as liposomes and microcapsules.

The formulations can be sterilized by numerous means, includingfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile medium justprior to use.

The antibody molecule may be suitable for and may be used in thetreatment or prevention of a disease. Accordingly, in some embodiments,an antibody molecule according to the invention may be used in a methodof treating and/or preventing a medical condition such as a disorder ordisease. Similarly, the antibody molecule of the present invention canbe used in the treatment of a disease. The disease to be treated orprevented may be a proliferative disease. Such a proliferative diseasemay preferably be tumor or cancer. Due to the ability of the antibodymolecule of the invention to bind PMSA, this antibody molecule can beused to treat cancer that consists of cells that express PSMA, such asprimary and metastatic prostate cancer cells (cf. , and theneovasculature of a wide spectrum of malignant neoplasms such asconventional (clear cell) renal carcinoma, transitional cell carcinomaof the urinary bladder, testicular embryonal carcinoma, colonicadenocarcinoma, neuroendocrine carcinoma, glioblastoma multiforme,malignant melanoma, pancreatic duct carcinoma, non-small cell lungcarcinoma, soft tissue sarcoma, and breast carcinoma (see in thiscontext, Chang SS et al. Five different anti prostate specific membraneantigen (PSMA) antibodies confirm PSMA expression in tumor associatedneovasculature. Cancer Res 1999; 59:3192). Thus, the antibody moleculeof the invention may in one aspect be used in the treatment of a solidcancer, such as prostate cancer, colon cancer, mammary cancer,pancreatic cancer or glioblastoma. However, due to the surprisingfinding that the antibody of the invention also binds to squamous cells,the antibody molecule of the invention may in another aspect bepreferably also be used in the treatment of squamous cell carcinoma ofdifferent origins (including but not limited to): carcinoma of the skin,head and neck, esophagus, lung and cervix uteri.

The subject to be treated with the fusion protein can be a human ornon-human animal. Such an animal is preferably a mammal, for instance ahuman, pig, cattle, rabbit, mouse, rat, primate, goat, sheep, chicken,or horse, most preferably a human.

The antibody molecule of the invention may also be used in the diagnosisof a disease, such as a disease as described herein. The antibodymolecule may for this purpose be labeled with a suitable detectablesignaling label. Such a labeled antibody molecule may permit detectionor quantitation of PSMA level or cancer such as prostate cancer, coloncancer, mammary cancer, pancreatic cancer or glioblastoma squamous cellcarcinoma as well as squamous cell carcinomas of different origin aslisted above in a sample or subject. When designated for in vivo use,said detectable signaling label is preferably detectable in vivo.

The labelled antibody molecule may be used in an immune- imagingtechnique. The detectable signaling label may then be selected, forinstance, based on the immuno-imaging technique employed for thediagnosis, for example, gamma-emitting radionuclide (or gamma-emitter)in case of gamma camera-imaging technique/SPECT, metal or positronemitter in case of MRI or PET imaging techniques, respectively. In thisregard, one or more detectable signaling labels of the disclosureinclude gamma camera-imageable agents, PET-imageable agents andMRI-imageable agents, such as, radionuclides, fluorescers, fluorogens,chromophores, chromogens, phosphorescers, chemiluminescers andbioluminescers.

A suitable detectable signaling label may be a radionuclide. Saidradionuclide may selected from the group consisting of ³H, ¹⁴C, ³⁵S,⁹⁹Tc, ¹²³1, ¹²⁵1, ¹³¹I, ^(m)In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr and ²⁰¹Tl.

A suitable detectable signaling label may also be fluorophore orfluorogen. Said fluorophore or fluorogen may be selected from the groupconsisting of fluorescein, rhodamine, dansyl, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, fluoresceinderivative, Oregon Green, Rhodamine Green, Rhodol Green or Texas Red.

The labelled antibody molecule may be coupled either directly orindirectly to a detectable signaling label. For example, the antibodymolecule may be coupled either directly (e.g. via tyrosine residues ofthe antibody molecule) or indirectly (e.g. via a linker--as a metalchelating agent) to a detectable signaling label. In some otherembodiments, the antibody molecule may be coupled to a molecule that isable to be coupled (either in vitro or in vivo) to the detectablesignaling label at the time and place of use.

A detectable signaling label may be bound to the antibody moleculethrough one or more diethylenetriaminepentaacetic acid (DTPA) residuesthat are coupled to the antibody molecule.

Also contemplated by the invention is an in vitro method of detecting ordiagnosing a disease defined herein. Such a method may comprisecontacting a sample obtained from a subject with a preferably labelledantibody molecule of the invention. The sample may be a blood, urine orcerebrospinal fluid sample, but may preferably be tissue sample or abiopsy sample. The disease to be detected or diagnosed is preferablyprostate cancer, colon cancer, mammary cancer, pancreatic cancer,glioblastoma or squamous cell carcinoma, most preferably squamous cellcarcinoma.

The present invention is further characterized by the following items

Item 1. An antibody molecule or an antigen-binding fragment thereof,capable of binding to human prostate specific membrane antigen (PSMA),comprising: (i) a heavy chain variable domain comprising the CDRH1region set forth in SEQ ID NO: 03 (GFTFSDFYMY), the CDRH2 region setforth in SEQ ID NO: 04 (TISDGGGYTSYPDSVKG), and the CDRH3 region setforth in SEQ ID NO: 05 (GLWLRDALDY) or comprising a CDRH1, CDRH2 orCDRH3 sequence having at least 75% sequence identity or at least 80%sequence identity with SEQ ID NO: 03, SEQ ID NO: 04, or SEQ ID NO: 05;and (ii) a light chain variable domain comprising the CDRL1 region setforth in SEQ ID NO: 06 (SASSSISSNYLH), the CDRL2 region set forth in SEQID NO: 07 (RTSNLAS), and the CDRL3 region set forth in SEQ ID NO: 08(QQGSYIPFT) or comprising a CDRL1, CDRL2 or CDRL3 sequence having atleast 75% sequence identity or at least 80% sequence identity with SEQID NO: 06, SEQ ID NO: 07, or SEQ ID NO: 08.

Item 2. The antibody molecule or antigen binding fragment thereof ofitem 1, wherein the heavy chain variable region comprises the amino acidsequence having a sequence identity of at least 90% to the amino acidsequence set forth in SEQ ID NO: 01 or 09.

Item 3. The antibody molecule or antigen binding fragment thereof ofitem 1 or 2, wherein the light chain variable region comprises the aminoacid sequence having a sequence identity of at least 90% to the aminoacid sequence set forth in SEQ ID NO: 02 or 10.

Item 4. The antibody molecule or antigen binding fragment thereof of anyone of the preceding items, wherein the antibody is selected from thegroup consisting of a scFv, a univalent antibody lacking a hinge region,a minibody, a Fab fragment, a Fab′ fragment, a F(ab′)₂ fragment, and awhole antibody.

Item 5. The antibody molecule or antigen binding fragment thereof of anyone of the preceding items comprising a human IgG constant domain.

Item 6. The antibody molecule or antigen binding fragment thereof of anyone of the preceding items comprising a CH1 domain.

Item 7. The antibody molecule or antigen binding fragment thereof of anyone of the preceding items comprising an Fc region.

Item 8. The antibody molecule or antigen binding fragment thereof of anyone of the preceding items, wherein the antibody has antibody-dependentcell mediated cytotoxicity (ADCC) effector function.

Item 9. The antibody molecule or antigen binding fragment thereof ofitem 8 having enhanced affinity to the FcγRIIIa receptor or has enhancedADCC effector function as compared to the parent antibody.

Item 10. The antibody molecule or antigen binding fragment thereof ofany one of the preceding items comprising a heavy chain and a lightchain and at least one amino acid substitution in the constant regionrelative to a parent antibody, wherein said at least one amino acidsubstitution comprises the amino acid substitutions S239D and I332E,wherein the positional numbering is according to the EU index.

Item 11. An antibody molecule or an antigen-binding fragment thereof,capable of binding to human PSMA that is able to compete with thebinding of an antibody molecule or antigen-binding fragment thereof ofitem 1 to human PSMA.

Item 12. The antibody molecule or antigen binding fragment thereof ofany one of the preceding items, wherein the antibody molecule orantigen-binding fragment thereof does not compete with the binding ofJ591 to human PSMA.

Item 13. The antibody molecule or antigen binding fragment thereof ofany one of the preceding items, wherein the antibody molecule orantigen-binding fragment thereof has a reduced induction of antigenshift when binding to PSMA than J591.

Item 14. The antibody molecule or antigen binding fragment thereof ofitem 13, wherein the antigen shift is induced in PMSA-transfected Sp2/0cells.

Item 15. The antibody molecule or antigen binding fragment thereof ofany one of the preceding claims, wherein the antibody molecule orantigen-binding fragment thereof further binds to squamous cellcarcinoma (SCC) cells.

Item 16. A bispecific antibody molecule comprising (i) a variable regioncomprising a heavy chain variable domain and a light chain variabledomain as defined in any one of the preceding items, wherein saidvariable region comprises a first binding site capable of binding tohuman prostate specific membrane antigen (PSMA); and (ii) a heavy chainvariable region and a light chain variable region of an antibodymolecule comprising a second binding site.

Item 17. The antibody molecule of item 16, wherein the first bindingsite and the second binding site bind to different binding partners.

Item 18. The antibody molecule of any item 16 or 17, wherein the firstor second binding site binds to a T cell or natural killer (NK) cellspecific receptor molecule.

Item 19. The antibody molecule of any one of items 16 to 18, wherein theT-cell-or NK cell specific receptor molecule is one of CD3, T cellreceptor (TCR), CD28, CD16, NKG2D, Ox40, 4-1 BB, CD2, CD5, PD-1 andCD95.

Item 20. The antibody molecule of any one of item 19, wherein the TCR isTCR (alpha/beta) or TCR (gamma/delta).

Item 21. The antibody molecule of item 19, wherein the T-cell- or NKcell specific receptor molecule is CD3.

Item 22. The antibody molecule of item 21, wherein the heavy chainvariable region and a light chain variable region of an antibodymolecule comprising a second binding site is the heavy chain variableregion and a light chain variable region of OKT3 or UCHT1.

Item 23. The antibody molecule of any one of items 15 to 21, wherein (i)the first binding site is comprised in a Fab fragment and the secondbinding site is comprised in a scFv fragment; or (ii) the first bindingsite is comprised in a single chain Fv fragment and the second bindingsite is comprised in a Fab fragment.

Item 24. The antibody molecule of item 23, wherein the Fab fragment andthe single chain Fv fragment are linked via a CH2 domain and/or a CH3domain.

Item 25. The antibody molecule of item 24, wherein at least one aminoacid residue of the CH2 domain that is able to mediate binding to Fcreceptors is lacking or mutated.

Item 26. The antibody molecule of item 24 or 25, wherein one or moreamino acid residues of sequence positions 226, 228, and 229 is lackingor mutated (numbering of sequence positions according to the EU-index).

Item 27. The antibody molecule of any one of item 24 to 26 wherein theFab fragment is linked to the CH2 domain via the heavy chain CH1 and VHdomains of the Fab fragment or via the CL and VL light chain domains ofthe Fab fragment.

Item 28. The antibody molecule of item 27, wherein the heavy chaindomains of the Fab fragment or the light chain domains of the Fabfragment are arranged at the N-terminus of the polypeptide chain of theantibody molecule.

Item 29. The antibody molecule of any of the preceding items, whereinthe Fab fragment comprises a hinge region.

Item 30. The antibody molecule of any one of items 24 to 29, wherein theat least one amino acid residue of the CH2 domain that is able tomediate binding to Fc receptors is lacking or mutated, is selected fromthe group consisting of sequence position 230, 231, 232, 233, 234, 235,236, 237, 238, 265, 297, 327, and 330 (numbering of sequence positionsaccording to the EU-index).

Item 31. The antibody molecule of any one of items 24 to 30, wherein acysteine at one or both of positions 226 and 229 is replaced by adifferent amino acid.

Item 32. The antibody molecule of any one of items 24 to 31 comprising aFab fragment, a CH2 domain and a scFv fragment, wherein the Fab fragmentcomprises a hinge region.

Item 33. The antibody molecule of item 32, wherein the Fab fragment is aFab fragment of a humanized 10B3 antibody and/or wherein the scFvfragment comprises a heavy chain variable region and a light chainvariable region from OKT3 antibody.

Item 34. The antibody molecule of item 33, wherein the heavy chain ofthe antibody molecule has a sequence as set forth in SEQ ID NO: 12and/or wherein the light chain of the antibody molecule has a sequenceset forth in SEQ ID NO: 13.

Item 35. The antibody molecule of any one of items 24 to 31 comprising aFab fragment, a CH2 domain, a CH3 domain and a scFv fragment, whereinthe Fab fragment comprises a hinge region.

Item 36. The antibody molecule of item 35, wherein the Fab fragment is aFab fragment of a humanized 10B3 antibody and/or wherein the scFvfragment comprises a heavy chain variable region and a light chainvariable region from a humanized UCHT1 antibody.

Item 37. The antibody molecule of item 36, wherein the heavy chain ofthe antibody molecule has a sequence as set forth in SEQ ID NO: 11and/or wherein the light chain of the antibody molecule has a sequenceset forth in SEQ ID NO: 13.

Item 38. The antibody molecule of any one of items 35 to 37, wherein theantibody molecule is a tetrameric antibody molecule.

Item 39. The antibody molecule of any one of items 16 to 23, wherein theantibody molecule is a bispecific tandem single chain Fv, a bispecificFab₂, or a bispecific diabody.

Item 40. A pharmaceutical composition comprising an antibody molecule oran antigen-binding fragment thereof as defined in any of the precedingitems.

Item 41. An antibody molecule or an antigen-binding fragment thereof asdefined in any of items 1 to 39 for use in the diagnosis or treatment ofa disease.

Item 42. The antibody molecule or antigen-binding fragment thereof forthe use of item 41, where the disease is a proliferatory disease.

Item 43. The antibody molecule or antigen-binding fragment thereof forthe use of item 42, wherein the proliferatory disease is cancer.

Item 44. The antibody molecule or antigen-binding fragment thereof forthe use of item 43, wherein the cancer is prostate cancer, colorectalcancer, cancer of the stomach, lung carcinoma, osteosarcoma, mammarycancer, pancreatic cancer or glioblastoma.

Item 45. The antibody molecule or antigen-binding fragment thereof forthe use of item 43, wherein the cancer is squamous cell carcinoma.

Item 46. An in vitro method of diagnosing a disease comprisingcontacting a sample obtained from a subject with an antibody molecule oran antigen-binding fragment thereof as defined in any one of items 1 to39.

Item 47. The in vitro method of item 46, wherein the sample is a tissuesample or a biopsy sample.

Item 48. The in vitro method of item 46 or 47, wherein the disease iscancer, preferably prostate cancer, colorectal cancer, cancer of thestomach, lung carcinoma, osteosarcoma, mammary cancer, pancreaticcancer, glioblastoma or squamous cell carcinoma.

Item 49. A nucleic acid molecule encoding an antibody molecule or anantigen-binding fragment thereof as defined in any of items 1 to 39.

Item 50. A vector comprising the nucleic acid molecule of item 49.

Item 51. A host cell comprising a nucleic acid molecule of item 49 or avector of item 50.

Item 52. A method of producing an antibody molecule or anantigen-binding fragment thereof of any one of items 1 to 39, comprisingexpressing a nucleic acid encoding the antibody molecule underconditions allowing expression of the nucleic acid.

Item 53. The method of item 52 wherein the antibody molecule orantigen-binding fragment thereof is expressed in a host cell or acell-free system.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 01: heavy chain variable domain of murine 10B3 antibody(amino acid sequence).

SEQ ID NO: 02: light chain variable domain of murine 10B3 antibody(amino acid sequence).

SEQ ID NO: 03: CDRH1 of murine 10B3 antibody (amino acid sequence).

SEQ ID NO: 04: CDRH2 of murine 10B3 antibody (amino acid sequence).

SEQ ID NO: 05: CDRH3 of murine 10B3 antibody (amino acid sequence).

SEQ ID NO: 06: CDRL1 of murine 10B3 antibody (amino acid sequence).

SEQ ID NO: 07: CDRL2 of murine 10B3 antibody (amino acid sequence).

SEQ ID NO: 08: CDRL3 of murine 10B3 antibody (amino acid sequence).

SEQ ID NO: 09: heavy chain variable domain of humanized 10B3 antibody(amino acid sequence).

SEQ ID NO: 10: light chain variable domain of humanized 10B3 antibody(amino acid sequence).

SEQ ID NO: 11: heavy chain of the humanized h10B3 X humanized hUCHT1bispecific IgGsc format antibody molecule

SEQ ID NO: 12: heavy chain of the humanized h10B3 X murine OKT3bispecific Fabsc format antibody molecule

SEQ ID NO: 13: kappa light chain of humanized 10B3 antibody

SEQ ID NO: 14: illustrative example of a mutated CDRH1 of murine 10B3antibody (amino acid sequence) having at least 75% sequence identity toSEQ ID NO: 3.

SEQ ID NO: 15: amino acid sequence of the variable domain of the heavychain of the antibody J519.

SEQ ID NO: 16: the amino acid sequence of the variable domain of thelight chain of the antibody J519.

EXPERIMENTAL EXAMPLES

The invention is further illustrated by the following non-limitingExamples.

Example 1: Generation, Identification and Production of the 10B3Antibody

The antibody 10B3 was generated after immunization of female BALB/c micewith irradiated PSMA transfected Sp2/0 Ag14 cells (the Sp2/0-Ag14 cellswere obtained from ATCC where they are commercially available under thename ATCC® CRL-1581™). Four days after the last immunization spleencells were fused with the transfected Sp2/0 cells and cultured in HATselection medium. Supernatants of growing hybridoma cells were screenedfor production of PSMA antibodies by flow cytometry using PSMAtransfected- and untransfected Sp2/0 cells. After subcloning by limitingdilution a stable monoclonal hybridoma cell line was obtained. Forantibody production hybridoma cells were adapted to advanced DMEM medium(Gibco,Thermo Scientific, Waltham, MA, USA02451), supplemented with 1%FCS (Biochrom GmbH, Berlin, Germany) to avoid contamination with bovineIgG during purification. The antibody was isolated from cell culturesupernatants by a Protein-A affinity chromatography and the isotype ofthe purified antibody was identified as IgG2b/kappa (RapidMouse-Monoclonal Isotyping Kit, BioAssay Works, Ijamsville, MD, 21754).The sequence of the variable heavy chain (shown in SEQ ID NO: 1 and FIG.10A) and of the variable light chain shown (shown in SEQ ID NO: 2 andFIG. 10B) was determined by Alvedron GmbH, Freiburg, Germany.

Example 2: Generation of a Humanized 10B3 Antibody

The 10B3 antibody was humanized by grafting the CDR regions into thegermline sequences of the human variable κ light sequence IGKV3-20*02(this sequence is deposited in the IMGT/LIGM-database under accessionnumber L37729, see also Ichiyoshi Y., Zhou M., Casali P. A humananti-insulin IgG autoantibody apparently arises through clonal selectionfrom an insulin-specific ‘germ-line’ natural antibody template. Analysisby V gene segment reassortment and site-directed mutagenesis’ J.Immunol. 154(1):226-238 (1995) and the variable heavy chain sequenceIGHV3-11*06 (this sequence is deposited in the IMGT/LIGM-database underaccession number AF064919, see also Watson C.T., et al. Completehaplotype sequence of the human immunoglobulin heavy-chain variable,diversity, and joining genes and characterization of allelic andcopy-number variation. Am. J. Hum. Genet. 92(4):530-546 (2013). It isnoted here that the IMGT/LIGM-DB is the IMGT comprehensive database ofimmunoglobulin (IG) and T cell receptor (TR) nucleotide sequences fromhuman and other vertebrate species; see Nucleic Acids Res. 2006 Jan1;34(Database issue):D781-4.

In order to maintain the binding properties of the parental murineantibody, the following two back mutations were introduced into theframework region of the variable humans. In the variable domain of theheavy chain of the human germline of IGHV3-11*06 the serine at position49 was back-mutated to an alanine that is present in the murine antibody10B3 (see also FIG. 10C in which the alanine residue at position 49 ishighlighted in bold and italics). In the variable domain of the lightchain sequence of IGKV3-20*02 the phenylalanine at sequence position 72of the human germline sequence was back-mutated to a tyrosine residuethat is present at this sequence position in the murine antibody 10B3(see also FIG. 10D in which the tyrosine residue at position 72 ishighlighted in bold and italics).

Example 3: Production of Recombinant Antibody Molecules and Off-Target TCell Activation by Different PSMAxCD3 Antibodies

For construction of recombinant bispecific antibody molecules, thevariable domains of the PSMA antibodies J591 and 10B3 were fused tohuman constant regions and variable regions of the CD3 antibodies OKT3or UCHT1 in the following order. VL-CL for both, the Fabsc- andIgGsc-formats. Heavy chains were constructed as follows:VH-CH1-CH2mod-scFv(OKT3/UCHT1) for the Fabsc format,VH-CH1-CH2mod-CH3-scFv(OKT3/UCHT1) for the IgGsc-format (cf. also FIGS.1A and 1B). In these antibody molecules, the PMSA binding site ispresent as Fab fragment while the CD3 binding site is present as scFvfragment (cf. again FIGS. 1A and 1B). To abrogate FcR-binding,glycosylation sites and the formation of disulfide bonds the followingmodifications were introduced into the hinge region and the CH2 domainof the Fabsc format (EU-index): C226S; C229S; E233P; L234V; L235A;ΔG236; D265G; N297Q; A327Q; A330S (see in this respect alsoInternational patent application WO 2013/092001). Modifications of theIgGsc formats were identical except for the two cysteine mutations atsequence positions 226 and 229 in the hinge region that are lacking inthe IgGsc-format. The constructs were cloned in an expression vectorderived from pcDNA3.1 (InVitrogen, Thermo Fisher) and transfected intoSp2/0 cells by electroporation as also described in International patentapplication WO 2013/092001. Antibody molecules were purified from thesupernatants of transfected cells by affinity chromatography withkappaSelect (Fabsc) or Protein A (IGGsc) resins. Both affinity resinswere purchased from GE Health Care Freiburg, Germany.

For characterization of the off-target T cell activation PBMC wereincubated with the indicated antibody molecules of J519 and 10B3 in theabsence and presence of SKW6.4 bystander lymphoma cells. After 2 daysCD69 expression of CD4 T cells was analyzed by flow cytometry. To thisend the PMBCs were incubated with detection antibodies directed to CD4(FITC labeled clone HP2/6), CD8 (APC-labeled clone HIT8a) and CD69 (PElabeled clone FN50) to identify T cells expressing the activation markerCD69. Cells were analyzed using a FACS Canto II from BD Biosciences.

Conclusion: As shown in FIG. 2 (which shows the results for thebispecific antibody molecules containing the variable domains of theantibody J519), the Fabsc antibody molecule containing the scFv fragmentof the CD3 binding antibody UCHT1 induces T cell activation in theabsence of PSMA expressing cells (off-target T cell activation) whereasthe IgGsc antibody molecule that comprising the same target, i.e. PMSAbinding site, and effector antibody binding site (i.e. the scFv fragmentof the CD3 binding antibody UCHT1) does not.

Example 4: On-Target T Cell Activation With PSMAxCD3 Antibodies in theFabsc-Format

In FIGS. 3A-3C T cell activation was assessed by ³H thymidine uptake. InFIG. 3A off-target T cell activation is shown for comparison. In FIG. 3Clysis of PSMA expressing target cells by activated T cells isdemonstrated by an Xelligence cytotoxicity assay. For the ³H-thymidineuptake assay PBMCs (20⁵/well) were seeded in triplicates in 96 wellplates and incubated with various concentrations of bispecific antibodymolecules with (FIG. 3B) or without (FIG. 3A) irradiated (100 Gy) PSMAexpressing 22RV1 cells (10⁵/well). After 72 hours, cells were pulsed foranother 20 hours with ³H-thymidine (0.5 µCi/well) and harvested onfiltermats. The incorporated radioactivity was determined by liquidscintillation counting in a 2450 Microplate counter (Perkin Elmer).

For the Xelligence assay 50 µl of culture media were added to 96 wellE-plates (Roche) to determine background values. Subsequently, targetcells (22RV1) were seeded at a density of 40.000 cells per well. Overthe next 20-24 hours cells were allowed to adhere to the wells. Then,PBMCs, isolated by density gradient centrifugation, were added to thetarget cells. The effector to target ratio (E:T) was 5:1 and thebispecific antibody concentration was 1 µg/ml. The impedance wasmonitored every 15 minutes for several days as a measure for theviability of the adherent target cells.

Conclusion: The bispecific PSMAxCD3 antibody containing OKT3 (NP-CO) isslightly less effective in mediating ,,on target” T cell activation andtarget cell killing as the reagent containing UCHT1 (NP-CU).

Example 5: Multimerization and Aggregation of Different BispecificAntibody Formats

In FIG. 4A and FIG. 4B antibody molecules with FLT3xCD3-specificity arecompared (Fabsc- vs bscc-format), while in FIGS. 4C-F those antibodymolecules with PSMAxCD3-specificity are compared (Fabsc- vs-IgGsc-format). The gel filtration that was used to analyze themultimerization behavior was performed on Superdex S200 columns.

Conclusions: (i) As shown in FIG. 4B, antibodies within the bssc format(see FIG. 1C for the bispecific single chain format used in theexperiment of FIG. 4B) have a marked tendency to form multimers andaggregates. This tendency is much lower in the case of the Fabsc- andIGsc-formats (FIG. 4A, FIGS. 4C-F). (ii) The moderate amount ofmultimers formed by the Fabsc/IgGsc constructs is higher for antibodiescontaining OKT3 (FIGS. 4C, 4E) compared to those comprising UCHT1 (FIGS.4D, 4F). While the results shown in FIGS. 4C to 4F have been obtainedwith Fabsc/IgGsc molecules that contain the variable domains of theantibody J591 as PMSA binding binding site, the analogous behavior hasbeen observed for the respective Fabsc molecules of the antibody 10B3(data not shown).

Example 6: Binding of the PSMA-antibodies J591 and 10B3 toPSMA-Expressing Cells

Binding (FIG. 5A), lack of binding competition (FIG. 5B) and shift ofthe PSMA antigen upon antibody binding (FIG. 5C) was assessed by flowcytometry using PSMA-transfected Sp2/0 cells. To this end differentα-PSMA antibodies were incubated with these cells in 96-well plates for30-45 min at 4° C. Cells were then washed and incubated with aPE-conjugated goat-anti-mouse F(ab)₂ fragment (FIGS. 5A, 5C) (JacksonImmunoResearch) or a PE-goat-anti-human FC-γ specific fragment (JacksonImmunoResearch) (B). Cells were analyzed on a FACSCalibur (BDBiosciences). In FIG. 5B it is demonstrated that the chimeric (ch) PMSAbinding antibody J591, specifically detected by a goat anti humansecondary antibody, was out-competed by murine (mu) antibody J591 butnot the murine antibody 10B3, i.e. an antibody of the present invention.For the determination of antigen shift (FIG. 5C) PSMA expressing cellswere incubated with the indicated antibodies at the beginning of theexperiment and again after 24 hrs and before FACS analysis with asaturating amount (10 µg/ml) of the respective antibody. PSMA expressionby untreated cells served as a reference (100% PSMA expression)

Conclusions: (i) Binding of both antibodies, J591 and 10B3 iscomparable, (ii) the antibodies do not cross-compete with each other,that is they bind to different epitopes and (iii) antigen shift inducedby the 10B3 antibody is markedly reduced when compared to that exertedby the antibody J591.

Example 7: Cryostat Sections Stained With the PSMA Antibodies J591 and10B3

In FIGS. 6A, 6B a prostate carcinoma cell sample was stained with bothantibodies while in FIG. 6C and FIG. 6D a squamous cell carcinoma samplewas stained with both antibodies. In both experiments the staining wascarried out in parallel and using a polymer system from Zytomed, Berlin,Germany (POLHRP-100). Arrows indicate tumor stroma (Tu) and bloodvessels (Ve). Representative results from 9 of 10 prostate cancersamples and 7 of 10 squamous cell carcinoma samples are shown.

On a variety of different normal human tissues (obtained from BioCat,Heidelberg, Germany, T6234701-2) the staining pattern of the twoantibodies was identical with the exception of a faint reactivity of theantibody 10B3 with epithelial cells in the skin.

Conclusions: (i) staining of prostate carcinoma samples and normaltissue is comparable with both antibodies, (ii) in squamous cellcarcinoma samples the J5191 antibody stains only vascular cells whereasthe antibody 10B3 stains vascular cells (more extensively than J591) andthe tumor cells themselves.

Example 8: Binding of Humanized and Mouse 10B3 Antibodies

Bispecific Fabsc antibodies with PSMAxCD3 (10B3xOKT3)-specificitycontaining either the variable domains of the humanized, CDR-grafted(h10B3) or mouse (m10B3) antibody were incubated with PSMA-expressing22RV1 cells and analyzed by flow cytometry (FIG. 7 ). To this end, thecells were incubated with the indicated antibodies in 96 well plates for30-45 min at 4° C. Upon incubation cells were washed and incubated witha secondary PE-goat-anti-human FC-γ specific fragment (JacksonImmunoResearch). Cells were analyzed on a FACSCalibur (BD Biosciences).

Conclusion: Binding of the mouse and humanized 10B3 versions(incorporated as a Fab moiety of the variable domains of 10B3 within aFabsc-antibody molecule) to PSMA expressing cells is identical.

Example 9: Binding of Different PSMAxCD3 Antibodies to CD3

Jurkat cells were incubated with the antibody molecules (Fabsc (UCHT1) =a Fabsc molecule comprising the CD3 binding variable domains of theantibody UCHT1 and the variable domains of the antibody J591; IgGsc(UCHT1) = IgGsc molecule comprising the CD3 binding variable domains ofthe antibody UCHT1 and the variable domains of the antibody J591, Fabsc(OKT3) = a Fabsc molecule comprising the CD3 binding variable domains ofthe antibody OKT3 and the variable domains of the antibody J591, IgGsc(OKT3) = IgGsc molecule comprising the CD3 binding variable domains ofthe antibody OKT3 and the variable domains of the antibody J591) in 96well plates for 30-45 min at 4° C. Cells were then washed and incubatedwith a Biotin-goat-anti-human IgG, F(ab′)₂ fragment specific (JacksonImmunoResearch) and Streptavidin-PE (Life Technologies). Cells wereanalyzed on a FACSCalibur (BD Biosciences).

Conclusion: As shown in FIG. 8 , avidity to CD3 is highest for theFabsc-antibody containing UCHT1, lowest for that containing OKT3. Withinthe IgGsc format the UCHT1 construct loses - the OKT3 construct gainsavidity. Since the avidity to of the bispecific antibody molecules toCD3 is obviously solely dependent on the CD3 binding (site), it is to beassumed that the ranking of the avidity of bispecific antibody moleculesthat contain the binding site of the antibody 10B3 will be the same asdetermined here using the variable domains of the antibody J591 asrepresentative PMSA binding site (target binding site).

Example 10: Cytolytic Activity of the Different PSMA Antibodies

PSMA expressing 22RV1 prostate carcinoma cells were incubated with PBMCsand the indicated bispecific PSMAxCD3 antibody molecules (Fabsc (UCHT1)= a Fabsc molecule comprising the CD3 binding variable domains of theantibody UCHT1 and the variable domains of the antibody J591; IgGsc(UCHT1) = IgGsc molecule comprising the CD3 binding variable domains ofthe antibody UCHT1 and the variable domains of the antibody J591, Fabsc(OKT3) = a Fabsc molecule comprising the CD3 binding variable domains ofthe antibody OKT3 and the variable domains of the antibody J591, IgGsc(OKT3) = IgGsc molecule comprising the CD3 binding variable domains ofthe antibody OKT3 and the variable domains of the antibody J591) at aPBMC:target ration of 5:1. The viability of the adherent target cellswas assessed using an Xelligence system as described in Example 4. Theeffector to target ratio (E:T) was 5:1 and the concentration ofantibodies was set to 5 nM.

Representative results of one out of four different experiments withPBMCs of different healthy volunteers are shown in FIG. 9 .

Conclusion: The ranking of lytic activity is: Fabsc (UCHT1) ≈ IgGsc(UCHT1) > Fabsc (OKT3) > IgGsc (OKT3). Since the lytic activity of thebispecific antibody molecules is dependent on the CD3 binding (site), itis to be assumed that the ranking of the lytic activity of bispecificantibody molecules that contain the binding site of the antibody 10B3will be the same as determined here using the variable domains of theantibody J591 as representative PMSA binding site (target binding site).

Example 11: Therapeutic Effect of PSMAxCD3 IgGsc (CC-1) in Vitro

PSMAxCD3 (h10B3xUCHT1)-IgGsc (CC-1) bispecific antibody of the inventionand a control bispecific antibody (NG2xCD3) were incubated with orwithout tumor cells (22Rv1 cells, human prostate carcinoma cells; seeSramkoski RM et al. In Vitro Cell Dev Biol Anim. 1999Jul-Aug;35(7):403-9.). CD4 and CD8 T cell activation was analyzed byFACS using CD69 and CD25 as cell surface markers after three days ofincubation. Both T cell types were activated (FIG. 15A), and interferongamma levels (FIG. 15B) and T cell proliferation increased (FIG. 15C).Chromium release assay (after 20 h E:T ratio 10:1) and FACS (over 72 h,E:T ration 1:1) furthermore showed a strong lysis of tumor cells usingCC-1 (FIGS. 15D and E). Treatment with CC-1 of the invention alsoinhibited tumor growth in vitro. At an E:T ratio of 2:1, tumor cellgrowth in the presence of CC-1 is significantly impaired as analyzedwith a Xelligence system (FIG. 15F left) and by visual inspection usinga microscope (FIG. 15F right).

Conclusion: CC-1 induces a tumor cell restricted activation of T-cellsand production of cytokines resulting in T cell proliferation andanti-tumor activity.

Example 12: Therapeutic Effect of PSMAxCD3 IgGsc (CC-1) in Vivo

Next, CC-1 (the IgGsc bispecific antibody of the invention) was testedfor anti-tumor activity in vivo in a mouse model. 1,5x10⁶ 22Rv1 cellswere injected into NSG (NOD scid gamma, (NOD.Cg-Prkdc^(scid)Il2rg^(tm1Wjl)/SzJ)) mice intravenously (n=4/group). After three days3x106 human PBMC were injected and on day 3 and 5 10 µg CC-1 or PBS asnegative control. After 8 days metastasis formation in the lung wasanalyzed using FACS. A significant reduction of the number of formedmetastases was found in the CC-1 treated group (FIG. 16A). Then, NSGmice (n=3/group) were injected with 2x10⁷ PBMC per mouse and treatedwith “supratherapeutic” CC-1 doses at 2x20 µg in an interval of 4 daysand a control antibody (UCHT1 as positive control, which activates Tcells unspecifically) or PBS as negative control. IFNgamma release andbodyweight as a marker of autoimmune activity was analyzed (FIG. 16B).CC-1 compared to UCHT1 did not induce IFNgamma production, and noreduction of bodyweight in CC-1 treated mice was observed, contrarily tothe UCHT1 treated group. In another experiment NOS/SCID mice wereinjected with 22Rv1 cells (PSMA prositive tumor cells) or with DU145cells (PSMA negative human prostate tumor cells) and after establishmentof tumors after 35 days, mice were treated with a radioisotope labeledCC-1 antibody (50 µCi) and sacrificed after 24 h to measureradioactivity in different organs. Only in tumors of 22Rv1 treated micea significant increase of radioactivity could be observed (FIG. 16C). Totest the difference of serum half-life of bispecific antibodies of theBiTE or IgGsc format of the invention, Balb/C mice were injected with 50µg of either CC-1 (IgGsc format) or the BiTE format bispecific PSMAxCD3antibody. Serum concentration was measured over time. Bispecificantibodies of the BiTE format were not detectable after 2-4 h ofinjection, whereas CC-1 was still detectable in the serum after 24 h(FIG. 16D). Therefore, the IgGsc format of the invention providesincreased serum stability compared to other bispecific antibody formats.

Conclusion: CC-1 suppresses tumor growth in vivo without inducing anyunspecific immune responses. CC-1 targets specifically PSMA expressingtumor tissue and has compared with other bispecific formats an increasedserum half-life.

Example 13: Comparison of UCHT1 and OKT3

To further elucidate the herein shown supremacy of UCHT1 over OKT3 asCD3 binding site in the IgGsc bispecific antibody format (see above),Fab and IgG versions of both monospecific antibodies were tested incomparison. OKT3 and UCHT1 were purified by protein A affinitychromatography. Fab fragments were generated by pepsin digestionfollowed by reduction and modification of hinge region disulfide bondsas previously described (Jung et al. Target cell induced T cellactivation with bi- and trispecific antibody fragments. Eur J Immunol1991; 21,2431-2435). Fab fragments were purified by size exclusionchromatography on a Superdex S200 column.

Then CD3 expressing Jurkat cells were incubated with increasingconcentrations of the indicated antibodies, a biotin labelled detectionantibody (anti-human Fab) followed by PE conjugated streptavidin.Samples were then analysed by flow cytometry (table 1). Concentrationsat which half maximal binding was observed are indicated (nM).

Table 1 Avidity of Fab and IgG Versions of anti-CD3 antibodies IgG FabOKT3 0.3 nM 23.7 nM UCHT1 0.6 nM 0.6 nM

Conclusion

OKT3 loses avidity if used as a univalent Fab fragment rather than abivalent intact IgG molecule. In contrast the avidity of the UCHT1fragment does not change. Without being bound to a theory, this pointsto a univalent binding of the intact UCHT1 antibody, the avidity ofwhich is comparable to the bivalently binding OKT3 antibody. The markeddifference in binding of the univalent Fab-fragments in favour of UCHT1explains the corresponding difference in binding of the two antibodieswithin the Fabsc-format (FIG. 8 , example 9). In this format theantibodies are present as a monovalent single chain molecule attached tothe C-terminus of a targeting antibody (FIGS. 1A-1C). Univalent bindingof UCHT1 also explains why this antibody loses- whereas OKT3 gainsavidity if present within the IgGsc-rather than the Fabsc-format (FIGS.1A-1C and FIG. 8 , example 9).

Although in the IgG-format the avidity of the UCHT1 containing molecule(IgGsc-UCHT1) is comparable or even slightly lower than that ofIgGsc-OKT3, the activity of IgGsc-UCHT1 against tumor cells is markedly-and unexpectedly- higher (FIG. 9 , example 10).

In summary IgGsc-UCHT1 is a format with optimized properties combininglow off target activation (FIG. 2 , example 3) with optimal lyticactivity against tumor cells (FIG. 9 , example 10).

Example 14: UCHT1 Is Effective in Other Bispecific IgGsc FormatAntibodies

The preferred use of UCHT1 as anti-CD3 antibody in an IgGsc basedantibody format was further tested for other non-PSMA specificantibodies. Irradiated M21 melanoma cells expressing the ganglioside GD2as well as an O-acetylated form of this antigen (oaGD2) were incubatedwith peripheral blood mononuclear cells (PBMC) isolated from theperipheral blood of normal human donors and bispecific IgGsc antibodieswith the indicated specificities. Parental monospecific antibodies usedwithin these constructs were hu14.18 (anti-GD2), 8B6 (anti-oaGD2), J591(anti-PSMA) and UCHT1 (anti-CD3), respectively. After 3 days T-cellactivation was assesssed using a ³H-thymidine incorporation assay.Further, M21 cells were incubated with PBMC and the indicated bispecificIgGsc antibodies (50 nM). Tumor cell growth was then monitored using anXelligence system. A bispecific IgGsc antibody with an unrelated targetspecificity (MOPC) was used as a control.

Conclusion

In the presence of tumor cells expressing GD2 and oaGD2 and bispecificantibodies targeting these antigens effective activation of T cellswithin the PBMC population was observed. A control antibody targetingPSMA was ineffective, since M21 do not express PSMA (FIG. 17A).

Bispecific IgGsc antibodies directed to GD2 or oaGD2 as targetspecificity and to CD3 as effector specificity effectively kill tumorcells expressing these antigens (FIG. 17B). The specific GD2- and CD3antibodies used are listed in the legend to FIG. 17A.

The invention illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by exemplary embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

What is claimed is:
 1. A tetravalent and homodimeric bispecific antibodymolecule comprising in each monomer: i) an N-terminal Fab fragmentcomprising a variable region comprising a heavy chain variable domainand a light chain variable domain, wherein said variable regioncomprises a first binding site capable of binding to an antigen; ii) aC-terminal scFv fragment, comprising a heavy chain variable region and alight chain variable region of humanized UCHT1, and wherein (i) and (ii)are connected by a CH2 and CH3 domain.
 2. The tetravalent bispecificantibody molecule of claim 1, wherein at least one amino acid residue ofthe CH2 domain that is able to mediate binding to Fc receptors islacking or mutated.
 3. The tetravalent bispecific antibody molecule ofclaim 1, wherein the Fab fragment is not a Fab fragment of a non-humanized, chimerized or humanized 10B3 or J591 antibody, preferablywherein the first binding site is not capable of binding to PSMA.
 4. Thetetravalent and homodimeric bispecific antibody molecule of claim 1,wherein the heavy chain variable region and the light chain variableregion of humanized UCHT1 comprises the sequence of UCHT1 as shown inSEQ ID NO: 11, starting with the amino acid sequence DIQMT (SEQ ID NO:17) and ending with VTVSS (SEQ ID NO: 18).
 5. The tetravalent bispecificantibody molecule of claim 1, wherein the Fab fragment binds to a tumorassociated antigen.